casein trapped silica nanospheres for controlled drug release

Journal Pre-proof pH-responsive hybrid hydrogels: Chondroitin sulfate/casein trapped silica nanospheres for controlled drug release

Andressa Renatta Simão, Vanessa H. Fragal, Antônia Millena de Oliveira Lima, Michelly Cristina Galdioli Pellá, Francielle P. Garcia, Celso V. Nakamura, Elias B. Tambourgi, Adley F. Rubira PII:

S0141-8130(19)40229-8

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.093

Reference:

BIOMAC 14397

To appear in:

International Journal of Biological Macromolecules

Received date:

11 December 2019

Revised date:

9 January 2020

Accepted date:

9 January 2020

Please cite this article as: A.R. Simão, V.H. Fragal, A.M. de Oliveira Lima, et al., pH-responsive hybrid hydrogels: Chondroitin sulfate/casein trapped silica nanospheres for controlled drug release, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.01.093

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© 2020 Published by Elsevier.

Journal Pre-proof

pH-responsive hybrid hydrogels: chondroitin sulfate/casein trapped silica nanospheres for controlled drug release Andressa Renatta Simãoa, Vanessa H. Fragala,b*, Antônia Millena de Oliveira Limaa, Michelly Cristina Galdioli Pelláa, Francielle P. Garcia,c Celso V. Nakamurac,d, Elias B.

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Tambourgib and Adley F. Rubiraa*

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a

Departamento de Química, Universidade Estadual de Maringá, Avenida

University of Campinas, Faculty of Chemical Engineering, Cidade

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b

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Colombo 5790, CEP: 87020-900, Maringá, Paraná, Brazil.

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Universitária Zeferino Vaz, Campinas, SP, Brazil. c

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Departamento de Ciências Básicas da Saúde, Universidade Estadual de

Maringá, Av. Colombo, 5790, CEP 87020-900, Maringá, Paraná, Brazil.

Programa de Pós-Graduação em Ciências Farmacêuticas, Departamento de

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Ciências Básicas da Saúde, Universidade Estadual de Maringá, Av. Colombo, 5790, CEP 87020-900, Maringá, Paraná, Brazil. *

Corresponding authors‘ e-mails: Vanessa H. Fragal ([email protected], Phone

55 44 3011-3687) Adley F. Rubira ([email protected], Phone 55 44 3011 3687)

Abstract In this study, the materials were synthesized by chemically crosslinking chondroitin sulfate (CS), casein (CAS), and silica nanospheres (SiO2), creating a highly crosslinked network. The hydrogel release profile was adaptable (that is, it could be

Journal Pre-proof faster or slower as needed) simply by changing the polymeric proportion. The incorporation of 5% of silica nanospheres, in mass, for all CAS/CS matrices promoted a better-controlled and sustained release of L-dopa, focusing on the matrix based on 70% of CAS, 30% of CS and 5% of silica, whose L-dopa release lasted for 87 h. Besides, hydrogels are cytocompatible. These new hydrogels can be considered highly attractive materials to be used for controlled and sustained drug release purposes, as well as

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scaffolds and wound dressing systems.

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KEYWORDS: casein; chondroitin sulfate; Stöber‘s silica; drug delivery device; L-

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dopa; Parkinson's disease.

1. Introduction

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Parkinson‘s disease, which affects 1 out of 100 people, is caused by insufficient

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production of dopamine at the dopamine neurons, leading to a neurodegenerative disorder that affects mainly a person‘s motor skills [1-3].

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L-dopa (3,4-dihydroxyphenylalanine) is a precursor to several neurotransmitters, especially dopamine. Its administration in individuals affected by Parkinson‘s disease started to be performed in 1961 [4], however, the L-dopa release must be controlled [5], because above therapeutic level, it leads to side effects such as vomiting and orthostatic hypotension [6]. Nowadays, several kinds of systems are used for a more efficient and controlled release of drugs. Double-layer materials [7], mesoporous inorganic materials [8], and polymeric materials (such as hydrogels) [9] are good examples to be mentioned, as well as hydrogel-based systems, which are interesting because of their adjustable drug release rate, maximizing the therapeutic effects [10].

Journal Pre-proof Hydrogels are highly-porous tridimensional materials, chemical or physically crosslinked [11, 12]. They have the ability to absorb water due to their thermodynamic affinity with solvents, [13], and, due to their porous structure, they can be used as drug carriers [14]. Another hydrogel interesting property, regarding drug delivery systems, is the ability to respond to different kinds of stimuli, being those stimuli internal and/or external. This class of material is called smart materials and the kind of stimulus they

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respond to directly influences their biological application as drug delivery systems [15].

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Hydrogels were the first kind of material to be directly used in the human body

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[16]. Regarding their composition, the selection of polymers to be used in the hydrogel

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synthesis is highly important once biocompatibility is a crucial factor to living

cytocompatible.

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organisms. Thus, the chosen polymeric matrices must be non-toxic, biodegradable, and

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Among the most common natural polymers used in hydrogels synthesis, there are the polysaccharides, which can be obtained from several sources like microbes,

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animals, and vegetables [17]. Hydrogels polysaccharide-based show properties such as biocompatibility, biodegradability, and bioactivity, which make them promising materials for biomaterial purposes [18]. Although chitosan and alginate are the most common polymers applied in hydrogels synthesis [19, 20], the chondroitin sulfate (CS), which is an animal-sourced polymer, found mainly in cartilages and in the cell surface, is also interesting for these purposes. Chemically, it is formed by bonds between D-glucuronic acid and N-acetylD-galactosamine, and the presence of functional groups (such as -COO- and -SO3-) makes the CS an interesting polymer to be used in hydrogel formation [5, 21]. Once it is

Journal Pre-proof highly water-soluble, crosslinking and/or blending with other macromolecules are alternatives often applied. Proteins are another interesting source of natural polymer for hydrogels synthesis [22], leading to biomaterials with resistance, high tenacity and elasticity [23, 24]. In this context, casein (CAS), which is a natural phosphoprotein obtained from skim milk, can be highlighted. It is biocompatible, non-toxic, and highly hydrophilic due to COOH, NH2 and OH groups in the polymeric network, which is also important

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for chemical modification reactions [25].

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Thus, polysaccharide and protein-based hydrogels are attractive for tissue

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engineering and drug delivery purposes [22]. However, none of these polymers (CS and

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CAS) has double bonds to allow the formation of the covalent bonds (crosslinking) responsible for the tridimensional structure. Therefore, they can only be used for

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hydrogel synthesis after being submitted to chemical modification reactions which, in

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this work, were performed by vinylic reagents: CS was modified by glycidyl methacrylate (GMA) and CAS was modified by maleic acid (MA).

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The combination of hydrogels and inorganic particles leads to materials with interesting properties regarding drug delivery systems. Among these inorganic nanoparticles, silica nanospheres (SiO2) can be highlighted because of their essential characteristics such as optical properties, high surface area, biocompatibility, low density, low-toxicity, adsorption capacity, and encapsulation capacity, allowing their use as biosensors, enzymatic support, and drug delivery systems, for example [26, 27]. Their combination of hydrogel formulations leads to a class of hydrogels called hybrid hydrogels. In this way, the aim of this work was obtaining a hybrid hydrogel based on CS, CAS, and SiO2 nanoparticles; evaluate the effect of different amounts of polymer over

Journal Pre-proof the final matrices‘ properties, as well as to evaluate the material‘s ability to acting as an oral drug delivery system for L-dopa.

2. Experimental procedures

2.1. Materials Chondroitin sulfate - SC (CAS 9007-28-7) with Mw 20,000 g.mol-1 determined

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by GPC/SEC was gently donated by Solabia, Brazil. Santa Clara milk (UHT integral);

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sodium persulfate (Vetec, 99%). Hydrochloric acid (36.5-38.0%) and acetone (99.5%)

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were obtained from Synth. Pure acetic acid (97%), ethyl ether (97%), dichloromethane

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(99.5%), sodium hydroxide in beads and ammonium hydroxide (99.5%) were acquired from Fmaia. Maleic anhydride - MA (97%), phosphate buffered saline - PBS (97%),

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glycidyl methacrylate - GMA (97%), tetraethyl orthosilicate - TEOS (98%),

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polyvinylpyrrolidone - PVP (Sigma-Aldrich, K12-18, 10000 g.mol-1),absolute ethanol (≥ 99.5%), N,N,N',N'-Tetramethyl ethylenediamine-TEMED (Initiation, 99%), Vitamin

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B12 (98%) were all from Sigma Aldrich.

2.2. Casein‘s extraction from skim milk Casein was isolated according to the literature‘s methods [28, 29]. At first, 150 mL of distilled water was heated until 38 °C and then 50 mL of milk was poured into the water. Next, acetic acid (0.7 mL) was added dropwise to the emulsion until precipitation. The dispersion settled for 20 minutes. The supernatant was separated through decantation and the precipitate was mixed with 20 mL of ethanol under magnetic stirring. The dispersion was, then, centrifuged and washed with ether. The final product, named Casein (CAS), was separated and dried over room temperature.

Journal Pre-proof 2.3. Casein (CAS) chemical modification using maleic anhydride (MA) The chemical modification was performed by adding 1 g of CAS and 0.1 g of MA to 50 mL of phosphate-buffered saline (PBS; 0.1 M; pH 5.5) and then the mixture was stirred for 12 h, at 60 °C. Next, the modified CAS was precipitated in cold ketone, centrifuged, and then lyophilized. The final material was named CASMA.

2.4. Chondroitin sulfate (CS) chemical modification using glycidyl methacrylate

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(GMA)

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The CS chemical modification using GMA was performed according to the

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literature [30]. Initially, 8.32 g of CS were solubilized in 150 mL of distilled water (2 h,

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room temperature). The pH was adjusted to 3.5 and 2.7 mL of GMA was added. The modification reaction lasted for 24 h and the system was kept under magnetic stirring, at

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50 °C. Then, the final material was precipitated in propanone, washed for GMA excess

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removal, centrifuged, and lyophilized. It was named CSGMA.

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2.5. Synthesis of the silica nanospheres (SiO2) The SiO2 (Stöber‘s silica) synthesis was performed according to literature methods [31]. At first, 4.5 mL of ammonium hydroxide solution (NH4OH; 28%) was mixed to 50 mL of ethanol and 2 mL of distilled water. After one minute of stirring, 3 mL of tetraethyl orthosilicate (TEOS) was added and the reaction was carried on for 24 h, under room temperature. Next, the solution was centrifuged for 15 min, at 2 °C and 9500 rpm. The supernatant was discarded and the material was washed 3 times using and hydroalcoholic solution (1:1).

Journal Pre-proof 2.6. Chemical attack on the SiO2 surface In a round-bottomed flask, under magnetic stirring, 0.5 g of SiO2 was dispersed in 50 mL of distilled water. Then, 0.4 of polyvinylpyrrolidone (PVP) was added and the system was kept in reflux for 3 h, at 100 °C. After this period, the material was naturally cooled and, then 0.2 g of NaOH was added to the round-bottomed flask and the mixture was stirred for 40 minutes. The final material was centrifuged and the supernatant was discarded. After that, the material was washed 3 more times (twice using distilled water

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and once using ethanol), and centrifuged for 15 minutes, at 2 °C, 9500 rpm.

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2.7. Double bond (C=C) insertion to the SiO2 nanoparticles

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In a round-bottomed flask, 500 mg of the chemically attacked SiO2 was dispersed in 250 mL of toluene. Then, 3 mL of vinyltrimethoxysilane (VTS) was added

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to the suspension and it was kept under reflux, at 80 °C, for 6 h. After this period, the

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modified nanospheres were washed, first using dichloromethane and then ethanol,

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centrifuged and lyophilized (24 h, -55 °C). The final material was named V-SiO2.

2.8. Hydrogels‘ synthesis

Hydrogels were synthesized using the proportions of CSGMA, CASMA, and VSiO2 shown in Table 1. The polymers were solubilized in 2 mL of distilled water under mechanical stirring, at room temperature and argon (Ar) atmosphere for 10 min. Afterward, 50 mg of sodium persulfate (Na2S2O8) 3 drops of TEMED were added to the reaction medium. At the end of the synthesis, the samples were already analyzed regarding the analysis described below. The hydrogels were named according to the percentage of CASMA/CSGMA/V-SiO2 respectively.

Journal Pre-proof Table 1. Percentage, in mass, of casein (CAS) chemically modified by maleic anhydride (MA), chondroitin sulfate (CS) chemically modified by glycidyl methacrylate (GMA), and silica microspheres modified by vinyltrimethoxysilane (V) utilized for the hydrogel synthesis. Samples

CSGMA (%)

V-SiO2 (%)

30-70-0

30

70

0

30-70-2

30

70

2

30-70-5

30

30-70-10

30

70-30-0

70

70-30-2

70

70-30-5

70

70-30-10

70

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CASMA (%)

5

70

10

30

0

30

2

30

5

30

10

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70

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CASMA/CSGMA/V-SiO2

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3. Characterizations 3.1. Swelling behavior

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The swelling behavior was evaluated through the material immersion in PBS solutions, at pH 1.2; 7.4, and 8.4, simulating the stomach, blood, and intestine pHs, respectively. The samples were kept at 37 °C and stirred at 50 rpm (Shaker Novatécnica). At specific times, the samples were weighed, and water excess was carefully removed. The hydrogel swelling degree equilibrium (Weq) was determined by Equation (1), where Wt is the swollen hydrogel‘s weight at the time t, and Ws is the dry hydrogel weight [32]. To check the reproducibility, for each sample, the swelling experiments were performed in duplicate. (Eq 1)

Journal Pre-proof 3.2. Scanning Electronic Microscopy (SEM) For the SEM and mapping images analysis, the fully swollen hydrogels (as described in section 3.1) were immediately frozen and lyophilized to avoid morphology changes. These samples were fractured to get their internal portion exposed and then metalized to be analyzed in a Quanta 250 SEM instrument (Thermo Fischer

3.3. Transmission Electron Microscopy (TEM)

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Scientific/Philips), at 15 kV and current intensity of 30 μA.

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The samples SiO2 and V-SiO2 were ultrasound-dispersed in ethanol and drops of

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these dispersions were placed in grids (200 carbon film square mesh copper grids).

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After that, the samples were analyzed in a transmission electron microscope (JEM-1400

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JEOL, at 120 kV).

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3.4. Nuclear Magnetic Resonance (NMR 1H) The NMR spectra were obtained in a Varian spectrophotometer (model Mercury

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Plus BB, 300 MHz). For that, samples were separately solubilized in 1.0 mL of D2O with 0.05 % of sodium 3-(trimethylsilyl) propionate (TPS; 2,2,3,3-d4), as internal reference. The standard pulse sequence was used and the parameters were: pulse width equals 90° (pw = 90°) and recycle time of 30 s (d1).

3.5. Fourier Transform (FT) – Raman The samples CAS, CASMA, CS, and CSGMA were analyzed by an FTIR-Raman spectrophotometer (model Vertex 70v with module Ram II, Bruker, Germany). The Raman equipment consisted of a germanium detector refrigerated by liquid nitrogen, a Nd:YAG laser at the wavelength of 1064 nm, nominal potency ranging from 50 to 300

Journal Pre-proof mW. Each spectrum was obtained by a mean of 128 scans with a spectral resolution of 4 cm-1, from 4000 to 400 cm-1.

3.6. Mechanical properties Mechanical properties were performed by compressing the cylindrical-shaped hydrogels (d = 0.5 mm and h = 1.0 mm) to a 1.0 mm strain using a texture analyzer TAX T2i equipped with a 5 kg load cell. A diameter of a 0.5 mm circular probe was

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adjusted to descend to the hydrogel surface moving at 2 mm/s. The tests were

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performed in three repetitions using post-synthesis hydrogels with a surface area of 10 cm . The data generated by the equipment were force and displacement, which were

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(Eq 2)

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converted into stress (σ) and deformation (λ), according to Eq 2:

where σ is the compressive stress, F is the applied force, A is the sectional area of the

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probe that compresses the hydrogel to a depth of 1 mm and λ is an apparent deformation. The elastic modulus of compression (E) was determined from the linear slope of the line by Eq 2.

3.7. Cytotoxicity assays A suspension of Vero cells was obtained through the confluent monolayer trypsinization, in DMEM (Dulbecco's Modified Eagle's Medium) supplied with 10% of SBF (Simulated Body Fluid). The cells were collected and counted in a Neubauer chamber, being previously diluted until the concentration of 2.5 x 105 cells/mL.

Journal Pre-proof The cells were, then, distributed in a 96-well plate and incubated at 37 °C, in a 5% CO2 atmosphere, for 24 hours. Then, the cell growth medium was removed and the samples (5 mg/mL) were co-cultured with the cells, being the tests performed in triplicate, and incubated once again, for 72 hours, under the same previously described conditions. Next, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed (2 mg mL-1 of stock solution, 50 µL well-1). After 4 hours of incubation, the formazan crystals were solubilized in DMSO [33]. The amount

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of formazan per well was determined by an ELISA reader (Bio-Tek®, model Power

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Wave XS), at 570nm and the cellular growth inhibition percentage was calculated.

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3.8. Controlled release

For the drug release assays, 1% of L-dopa was added to the hydrogel (regarding

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its total mass). The controlled release was performed in a PBS buffer solution, at pHs

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1.2; 7.4, and 8.4, until the equilibrium. The system was kept under stirring (50 rpm) in a shaker (Shaker, model Novatécnica), at 37 °C. The amount of released drug was

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determined by a UV-Vis spectrophotometer (Genesys 10S Thermo Fisher Scientific), at 360 nm. To check the reproducibility, for each sample, the controlled release experiments were performed in duplicate.

Journal Pre-proof 4. Results and Discussion 4.1. Chemical modifications CS was modified according to previously published manuscripts [30, 34], being the modification confirmed by FT-Raman Figure 1(b), and NMR ¹H analysis Figure 1 (c, d, and e). The major product obtained from this chemical modification reaction (CSGMA), is shown in Figure 1(a), while presents the FT-Raman spectra for CS and CSGMA.

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As shown in Figure 1(b), it is possible to confirm the CS modification by the

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band placed at 1712 cm-1, characteristic of ester carbonyl groups (νC=O)[35], which are

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not naturally found in CS structure [36-38]. Also, in the NMR ¹H analysis Figure 1 (c,

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d, and e), the signals at δ 6.07 and δ 5.65 in the CSGMA spectra (hydrogens 1 and 1‘) confirm the presence of hydrogen atoms from vinylic carbons from GMA. The signal at

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δ 1.83 comes from methyl hydrogens (hydrogens 2), also from GMA [39]. This

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Raman results.

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additional evidence confirms the CS‘s modification by GMA, corroborating the FT-

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Figure 1. (a) CSGMA structure, (b) CS and CSGMA FT-Raman spectra, (c) CS, GMA and CSGMA NMR 1H spectra (d) and (e) CS, GMA and CSGMA NMR 1H spectra expansion or magnification.

The CAS chemical modification by MA occurred through a nucleophilic attack. The amino groups, mainly lysine residue, were responsible for attacking the MA‘s

Journal Pre-proof carbonyl group. Figure 2 (a) shows part of the modified CAS. The modifications were identified by FT-Raman and NMR H1. The FT-Raman spectra to CAS and CASMA, Figure 2 (b), show an intense band at 920 cm-1, characteristic of out-of-plane twisting [40] of a trans-disubstituted double bond (C=C) [35]. It indicates a conformational change (from cis to trans) due to the MA ring opening. The signal at δ 6.2 from the CASMA spectra (hydrogens a and a‘) confirm the presence of vinylic hydrogens in the CAS structure, due to the MA modification. This way, it is possible to affirm that the

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obtained by the FT-Raman analyses Raman [41].

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protein modification by MA was effective and it is in concordance with the results

Figure 2. (a) A piece from CASMA‘s structure, (b) CAS and CASMA FT-Raman spectra, and (c) CAS e CASMA NMR 1H spectra.

Figure 3(a) shows the stages of the V-SiO2 synthesis. At first, the nanospheres were attacked by NaOH to have their surface area increased, and to increase the degree of modification by VTS (third step; section 2.7) ) [42]. By the FT-Raman spectra, shown in Figure 3(b), it was possible to notice the arising of vibrational bands at 1635

Journal Pre-proof cm-1, due to the C=C stretching (ᵛC=C), at 1450 cm-1, due to the characteristic scissoring deformation of =CH2 bonds (ᵟCH2=), while rocking deformation (ᵖCH=) can be seen at 1280 cm-1. These bands confirm the presence of a vinyl group on the SiO2 nanospheres‘ surface [42, 43], and that the modification was effective. The TEM images referring to SiO2 nanoparticles‘ synthesis and modification are shown in Figure 3 (c). It is observed that, right after the synthesis, the nanospheres were well-defined and their diameter was about 250 nm, as indicated by Dynamic Light

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Scattering (DLS) assays (Figure 3 (f)). After the alkaline treatment, their surface

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became less homogeneous and erosion marks could be seen (Figure 3 (d)). The double

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bond attachment to the SiO2 nanospheres (V-SiO2) increased their diameter, easing the

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covalent bond formation between them and the hydrogel polymers (Figures (e) and

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(g)).

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Figure 3. (a) V-SiO2 reactional scheme, (b) SiO2 and V-SiO2 FT-Raman spectra. TEM images after (c) the Stöber‘s silica synthesis (SiO2) and (d) the ―SiO2‖ nanospheres after the NaOH treatment; (e) SiO2 nanospheres after the chemical modification by VTS (VSiO2). (f) DLS from the SiO2 nanospheres, and (g) DLS from the V-SiO2 nanospheres.

Journal Pre-proof After all the compounds modification reactions and the confirmation of their modification, it was possible to synthesize the chemical hydrogels. To better understand

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their synthesis, an illustrative scheme is presented in Figure 4.

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Figure 4. Illustration of the synthetic procedure used in the syntheses of

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CSGMA/CASMA/ and CSGMA/CASMA/V-SiO2 hydrogels, which can be used for drug delivery purposes. At first, all the reagents were dissolved in water; then, AR was

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inserted in the system for 10 minutes. Next, the radical initiator and the catalyst were

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added to the mixture. Once all the reagents got mixed, they were transferred to a syringe

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and dried in an oven for 10 minutes, at 50 °C.

The hydrogel formation is started by the homolytic breaking of the Na2S2O8 peroxide bond, catalyzed by TEMED. This step is important for the radical formation, responsible for the following crosslinking reactions. Once the sodium persulfate becomes a radical species, the contact with the polymers (CASAM, CSGMA e V-SiO2) starts the propagation step, in which the radical activates the double bond, and new radical species are formed. The reaction ends by combination or disproportion, producing hydrogel.

Journal Pre-proof 4.2. Morphology analysis Aiming to evaluate the polymeric distribution in the hydrogel, SEM with mapping by Energy Dispersive X-Ray analysis was performed (Figure 5 e Figures S1, S2 e S3). All the mappings indicated that only CAS elements, like phosphor (P), calcium (Ca), and nitrogen (N), are distributed in the hydrogel‘s exposed areas. However, it is important to point out that SiO2 nanospheres were also observed for the hydrogels in which they were present, and they were well-distributed in the matrices.

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These results confirm, qualitatively, a homogeneous polymeric distribution in

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hydrogels.

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Figure 5. Elemental mapping images for the hydrogel 30-70-10.

4.3. Mechanical properties The successful application of hydrogels as controlled drug delivery systems, which are often used for longer therapies, has a major contribution to their mechanical characteristics. In the event of failure or reduced mechanical strength, the integrity of the hydrogel under certain conditions is affected.

Journal Pre-proof The mechanical behavior of the samples was examined using the modulus of elasticity (E). E is obtained when the material undergoes elastic or non-permanent deformation. This means that when the gel is subjected to a compressive load, the deformation is accommodated by reorganizing the polymer chains within the gel. When the applied load is released, the currents return to their original settings. Elastic strain corresponds to the linear portion of the stress curve versus strain. The E was obtained from the linear slope (up to 20% of the strain) of the stress-

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strain curves (Figure 6), plotted according to Eq 2. Remembered, how bigger is the E

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more stress is required to create the same amount of strain. The E of the hydrogels

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ranged from 2.10-4- 7.10-4 MPa (Table S1). The E for matrix 30-70 hydrogels are higher

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compared to matrix 70-30. This fact may be related to the higher proportion of polysaccharide dispersed in the matrix, which decreases the hydrogel flexibility. This

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was expected because casein has high resistance, tenacity, and elasticity [23, 24].

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However, in the matrices the silica insertion did not significantly affect the E

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(except in the 30-70-2, where E decreased and in relation to the other 30-70 matrices).

Figure 6. Stress-strain curves of hydrogels with and without V-SiO : (a) matrix 30-70 2

and (b) 70-30.

Journal Pre-proof 4.4. Swelling behavior In Figure 7 (i), the swelling behavior for the matrices 30-70 and 70-30, at different pHs, is presented. All the 30-70 matrices were able to swollen (in at least 50%) at pH 1.2, more than for the other evaluated pHs. This behavior was expected due to the higher amount of negative charges in these hydrogels, once the 30-70 matrices have higher amounts of CS, which is an anionic polymer [44]. Thus, the higher amount of positive charges in the medium increases the attraction to the matrix, leading to a higher

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swelling at pH 1.2. For the other evaluated pHs (7.4 and 8.4), the swelling degree was

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inferior due to the smaller charge difference between the medium and the matrix.

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Therefore, as the chemical potential between the buffer solution and the hydrogel

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decreases, the mass increase is also inferior (even 2 times), when compared to the results from pH 1.2.

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On the other hand, for the matrices 70-30, the swelling degree was superior at

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pHs 7.4, and 8.4, when compared to pH 1.2. It might have happened due to CAS (higher amount of polymer in this matrix) being more cationic than CS, decreasing the negative

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charges in the hydrogel. Thus, a greater chemical potential difference is not observed between the hydrogel and the medium at pH 1.2, but at higher pH-values, like 8.4. In Figure 7 (i), it is possible to notice a higher swelling tendency for hydrogel 70-30 at pH 8.4 (about 4 times higher than the pos-formation hydrogel) than at pH 1.2 (where the hydrogel‘s mass is increased only about 2 times).

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Figure 7. Swollen hydrogels SEM images at pH 7.4 for the matrices (a) 30-70-0 (b) 3070-2; (c) 30-70-5; (d) 30-70-10; (e) 70-30-0; (f) 70-30-2; (g) 70-30-5; (h) 70-30-10; (i)

Journal Pre-proof Swelling degree at different pHs for the hydrogels 30-70 and 70-30, with varied silica amounts.

The pH influence over the pore morphology is also shown in Figure 7 and Figures S4 and S5. The combination of different amounts of modified CS, CAS, and SiO2 led to interesting behaviors regarding pore sizes. Casein is more hydrophilic than CS due to the several hydrophilic groups in its structure, while SiO2 is negatively

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charged [45]. It was expected that higher amounts of CAS would lead to bigger pores

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[46] once it is more hydrophilic than CS. However, it was not observed. It was noticed

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that higher amounts of CS led to bigger pores with low pore density, while higher

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amounts of CAS led to smaller pores, but presenting higher pore density. The observed behavior might have happened due to higher repulsion forces in

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the matrix 30-70, because of the group SO3- and the SiO2 particles in the matrix, leading

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to bigger pores to reach a repulsive equilibrium in the matrix. It is confirmed when analyzing the observed swelling behavior at pH 1.2, where the matrix‘s negative

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charges were attracted by the medium‘s positive charges, leading to higher pore expansion and, consequently, higher swelling values. On the other hand, the matrix 70-30, did not feel repulsive forces as intense as the ones observed for the matrix 30-70, once the amount of SO3- was smaller in the matrix 70-30. This way, at pH 1.2, there were attractions between the CS‘s and SiO2 negative charges and the medium, but the strongest interaction was reached at pH 8.4, where the medium‘s negative charge and the innumerable amount of CAS‘s positive group interacted. These results confirm the hydrogels‘ potential application as pH-responsive materials. It is possible due to their ability to respond to the environment they are

Journal Pre-proof exposed to, being their swelling process controlled by changing the polymers‘ amount. Regarding the L-dopa release, the most interesting matrix is 70-30 considering its main absorption occurs in the intestine [47]. Similar swelling results were found by Zhao and coworkers [48], whose methacrylate alginate-based hydrogels showed lower swelling ratios at pH 2.1 because of hydrogen-bonding between the groups COOH and OH from alginate, CONH from poly(N-isopropyl acrylamide), and -C-O- and OH from poly(ethylene glycol) methacrylate.

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However, at higher pH values (pH 7.4), the negative charge from the carboxyl group

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from alginate was capable of increasing the hydrogels‘ swelling capacity due to the

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electrostatic repulsion between the chains.

4.5. Controlled release

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Regarding the L-dopa controlled release, the analyses were performed at

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different pH values (1.2, 7.4, and 8.4) to simulate the environments to which the oral drug delivery system would be exposed to. In Figure 8 the matrices‘ release profile is

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shown, correlating the drug release over time, at different pH values (1.2, 7.4, and 8.4). The release profile and the different times required to reach the release equilibrium, confirm the materials‘ pH-responsiveness. This discussion will focus on pH 7.4, once it simulates blood pH, where the drug acts. For the matrices 30-70, at pH 7.4, hydrogel 30-70-5 showed the slowest release among the evaluated ones, taking 22 hours to release 50% of the loaded amount of drug (t50 = 22 h) and 41 hours to release 90% (t90 = 41 h). On the other hand, the matrix 30-70-10 only took 4 hours to release 50% of the drug, and 10 hours to release 90%. For the matrices 70-30, at pH 7.4 (Figure 8), the L-dopa release is even more controlled than the observed for the 30-70 matrices. The matrix containing 5% of V-

Journal Pre-proof SiO2 showed a slow-release (t50 = 13 h and t70 = 87 h) when compared to the one containing 2% of V-SiO2 (t50 = 8 h e t90 = 44 h). It confirms that, for both matrices (3070 and 70-30), the ideal percentage of V-SiO2 for improved drug release is equivalent to 5%. Drug release profiles of different materials can be evaluated by several models and methods [49]. In our study, the complex release profiles observed for these hybrid materials well-fit the model of Korsmeyer-Peppas and Weibull [50]; [51, 52]. At first,

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the release profiles were divided into two stages: the fast release early stage (until 60%)

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and a slower release stage. The initial stage was evaluated according to the model of

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Korsmeyer-Peppas, and the second stage, according to the model of Weibull. The

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evaluated parameters are shown in Table 2 (pH 7.4), Table S2 (pH 1.2) and Table S3 (pH 8.4).

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For both stages, the L-dopa release well-fit the models, once excellent R² values

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(R² > 0.95) were observed, as shown in Table 2. The diffusion mechanism exponent can be determined by the ‗n‘ value from the Korsmeyer-Peppas‘ equation, in which for

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cylindric materials, n=0.45 represents the Fickian diffusion; 0.450.89, super transport case II.

The hydrogels 30-70, with 0.5, and 10% of V-SiO2, presented values correspondent to anomalous transport, indicating that the drug-releasing process is partially influenced by the polymeric chain relaxation. However, the sample 30-70-2 led to Fickian diffusion. On the other hand, the drug release in the matrices 70-30 is governed by the super transport case II. It means that the higher amount of CAS leads to their increase of the hydrogel density, and, consequently, the increase of its rigidity. In this case, the

Journal Pre-proof initial release behavior is not affected by a possible chain relaxation. The matrix 70-305 has a zero-order drug release. That is, the release is as controlled as possible, being the drug release at a constant rate, represented by the kinetic release constant (k) [53]. Zeroorder release systems are often requested for the release of several kinds of drugs, including L-dopa. It should be highlighted that all the synthesized hydrogels presented low k values, ranging from 0.024 to 0.173 μg.h-1, inferring a controlled release in all the

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assays. However, the k value is directly influenced by the hydrogel‘s composition. For

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the 70-30 matrices, k ranged from 0.024 to 0.068 μg.h-1, which is much lower than the

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values observed for the 30-70 matrices, whose k values ranged from 0.094 to 0.173

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μg.h-1. Furthermore, even higher k values could be related to higher amounts of CS. Therefore, higher amounts of CAS led to more versatile hydrogels, which is strictly

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related to the protein‘s several amino acids, increasing the drug-matrix interaction.

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Moreover, CAS can also increase the matrix‘s tenacity. Thus, higher amounts of CAS combined with CS and V-SiO2 led to an interesting hybrid hydrogel for L-dopa‘s

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release, as well as to other drugs.

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Figure 8. L-dopa release from matrices 30-70 and 70-30, with different amounts of VSiO2, at pHs 1.2, 7.4, and 8.4.

Journal Pre-proof Table 2. Diffusion Exponents (n) and diffusion mechanisms determined by KorsmeyerPeppas‘ and Weibull‘s models for L-dopa‘s release from hydrogels 30-70 and 70-30, with different proportions of silica, at pH 7.4.

Initial Release Stage

0.82 ± 0.03

0.99

30-70-2

0.44 ± 0.01

0.98

30-70-5

0.87 ± 0.04

0.98

30-70-10

0.72 ± 0.05

0.97

70-30-0

0.97 ± 0.01

0.98

70-30-2

1.25 ± 0.01

0.99

70-30-5

0.89 ± 0.03

0.98

70-30-10

0.98 ± 0.05

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R2

0.094 ± 0.002

0.98

0.06 ± 0.002

0.98

0.122 ± 0.05

0.98

0.173 ± 0.01

0.98

0.041 ± 0.004

0.97

0.068 ± 0.006

0.99

0.046 ± 0.01

0.96

0.024 ± 0.008

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0.97

k (μg.h-1)

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30-70-0

Diffusion Mechanism Anomalous transport Fickian diffusion Anomalous transport Anomalous transport Super Transport Case II Super Transport Case II Transport case II Super Transport Case II

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R2

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N

Retarded Release Stage

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Samples CAS/CS/V-SiO2

To verify if these hydrogels could or could not be used as biodevices, cytotoxicity assays were performed (Figure 9). The desirable cell toxicity values range from 0% to 50% (CC50). Above 50%, the material is considered toxic and, below this value, its use is recommended [54, 55].

4.6. Cytotoxicity As indicated in Figure 9, the hydrogels did not reach the toxic levels. It is also possible to affirm that only a few changes in cell viability were observed for the hydrogels at 5 mg mL-1 concentration, once 70-30 reached 90% and 30-70, 80%. It was

Journal Pre-proof also noticed that higher amounts of V-SiO2 barely influenced the hydrogels‘ cell

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viability. Therefore, it is safe to affirm they are cytocompatible.

Figure 9. Cell viability from Vero cells determined by the MTT assay, after 72 hours of

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exposure to a hydrogel co-cultured with the cells with the concentration of 5 mg mL-1,

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for the matrices 30-70 and 70-30, with 0% or 10% of V-SiO2. The standard deviation

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5. Conclusion

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was obtained from three measures (n=3).

New hybrid hydrogels for biomedical applications were synthesized by the combination of silica nanospheres, protein casein (CAS), and polymer chondroitin sulfate (CS). The synthetic method allowed water stabilization in the hydrogels with well-aligned pores, especially for the ones whose CS levels were higher. The incorporation of 5% of silica nanospheres for all the proportions of CAS/CS led to an increased drug release control, being the matrix 70-30-5 the one with the best results regarding drug trapping and release, besides being the most compatible one. The in vitro cytotoxicity assays indicated no toxicity from the hydrogels, indicating that the hybrid hydrogels based on CAS-CS-V-SiO2 can be used as a multifunctional oral drug delivery system, accomplishing several of the required

Journal Pre-proof properties for biomedical application. In addition, the synthetic approach used in this work can be also applied for other efficient hybrid materials synthesis, for drug administration purposes. The evaluated hydrogels are also capable of releasing drugs other than L-dopa, and their structure allows their use as scaffolds, and wound-dressings as well, but further studies are necessary to assess how effective they would be for the mentioned possible uses.

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6. Acknowledgments

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ARS thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

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(CAPES-Brasil) for doctorate fellowships. A.F.R acknowledges the financial support

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given by CWPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

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7. References

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(CAPES-Brasil) and Fundação Araucária-Brasil.

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Author Statement

Andressa Renatta Simão: Methodology, Validation, Investigation, Formal

H.

Fragal:

Conceptualization,

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Visualization and Writing - Review & Editing

Methodology,

Investigation,

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Vanessa

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analysis and Writing - Original Draft

Antônia Millena de Oliveira Lima: Investigation and Writing - Original Draft

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Michelly Cristina Galdioli Pellá Writing - Original Draft and Writing -

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Review & Editing

Francielle P. Garcia: Investigation

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Celso V. Nakamura: Resources

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Elias B. Tambourgi - Supervision and Funding acquisition Adley F. Rubira- Supervision and Project administration

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Graphical abstract

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Journal Pre-proof Highlights -

Hybrid hydrogels were synthesized by combining silica nanospheres with chondroitin sulfate polymer and casein protein.

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Hybrid hydrogels with different percentages of silica was used for drug delivery of L-dopa.

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The incorporation of 5% of silica nanospheres in hybrid hydrogels promoted increased control and sustained release of L-dopa.

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All hybrid hydrogels obtained are biocompatible.

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Synthesized hybrid hydrogels can be effective in the administration of numerous

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drugs and in applications requiring the use of biomaterials.