In the present work, hybrid microgels based on chitosan and SiO2 nanoparticles (NPs) were synthesized. Both chitosan and the SiO2 NPs were submitted to chemical modification reactions to having vinyl groups incorporated into their structures.
Carbohydrate Polymers 239 (2020) 116236 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Chitosan hybrid microgels for oral drug delivery a, a T b Michelly Cristina Galdioli Pellá *, Andressa Renatta Simão , Michele Karoline Lima-Tenório , Ernandes Tenório-Netob, Débora Botura Scariotc, Celso Vataru Nakamurac, Adley Forti Rubiraa,* a b c Department of Chemistry, State University of Maringa, Av Colombo, 5790, CEP, 87020-900, Maringa, Parana, Brazil Department of Chemistry, State University of Ponta Grossa, Av Gen Carlos Cavalcanti, 4748, CEP 84030-900, Ponta Grossa, Parana, Brazil Department of Basic Science of Health, State University of Maringa, Av Colombo, 5790, CEP 87020-900, Maringa, Parana, Brazil A R T I C LE I N FO A B S T R A C T Keywords: Chemical modification SiO2 nanoparticles Glycidyl methacrylate In the present work, hybrid microgels based on chitosan and SiO2 nanoparticles (NPs) were synthesized Both chitosan and the SiO2 NPs were submitted to chemical modification reactions to having vinyl groups incorporated into their structures The microgels were synthesized by emulsion polymerization SEM analysis indicated a high dispersity of diameter for the microgels, ranging between (18.7 ± 12.3) μm for the samples without SiO2-VTS and (11.3 ± 8.07) μm for the microgels with SiO2-VTS The material showed pH-responsiveness, especially in acidic pHs The longest release lasted 45 and large amounts of drugs were released as soon as the material was added to the release medium It is interesting for oral drug delivery systems, especially for gastric wound treatment The fast release of high amounts of drugs promotes an immediate relief of the pain and the following controlled release allows the gradual recovery of the damaged area Introduction The development of devices with efficient controlled release behavior is a big challenge regarding gastrointestinal disorders (Ensign, Cone, & Hanes, 2012) Inorganic nanoparticles (Heneweer, Gendy, & Peñate-Medina, 2012), hydrogels (Langer & Peppas, 2003; Soares et al., 2016; Wang et al., 2013), microgels (Bysell, Månsson, Hansson, & Malmsten, 2011; Sivakumaran, Maitland, & Hoare, 2011), and nanogels (Wang et al., 2013) are examples of devices used as drug delivery systems Hydrogels are tridimensional devices, chemical or physically crosslinked (Ahmad, Rai, & Mahmood, 2016) This tridimensional structure allows the allocation and transport of bioactive molecules, like drugs (Ahmad et al., 2016) It minimizes or prevents the effect of different physiological environments over the drugs (Langer & Peppas, 2003) Hydrogels can be used in a macro, micro or nanoscale (Ahmad et al., 2016), being their size an important factor regarding the form in which the hydrogel will be administrated They can be orally administrated (Park, 1988), implanted (Cohn, Sosnik, & Garty, 2005) or injected in the body (Jeong, Bae, & Kim, 2000) Among their properties, these tridimensional gels can respond to several types of stimuli like pH, ionic strength, temperature, electromagnetic field (Grainger, 2013), etc They also can swell water, expanding their chains, which allows the release of bioactive agents ⁎ entrapped in their structure It makes hydrogels a minimally invasive device (Grainger, 2013) Synthetic and natural polymers are suitable for the obtention of hydrogels and microgels (Ahmad et al., 2016) However, when it comes to biological applications, natural polymers become more interesting because they are biocompatible, biodegradable, and non-toxic (Simão et al., 2020) Chitosan (CTS) is a polysaccharide widely used in the synthesis of hydrogels and microgels (Kang & Kim, 2010; Zhou et al., 2016) Due to the presence of amino-groups (-NH2) in its structure, chitosan is a positively charged polymer whose chains can be easily modified (Zeng, Fang, & Xu, 2004) This polymer is biocompatible, biodegradable, nontoxic (Zeng et al., 2004), and also shows antimicrobial activity (Xu et al., 2012) Furthermore, chitosan-based devices have been used for the delivery of drugs destined for the treatment of gastrointestinal disorders (Hejazi & Amiji, 2003) In the past years, drug delivery devices have been improved by the combination of polymeric devices (like microgels) and inorganic nanoparticles (Grainger, 2013; Lu, Zahedi, Forman, & Allen, 2014) These inorganic nanoparticles can be biocompatible, non-toxic, and bioabsorpt (Soares et al., 2016) They have been being combined with polymeric materials like, for example, aiming to improve the drug delivery system (Lu et al., 2014) One example of inorganic nanoparticle with attractive properties is Corresponding authors E-mail addresses: michellepella57@gmail.com (M.C Galdioli Pellá), afrubira@gmail.com (A.F Rubira) https://doi.org/10.1016/j.carbpol.2020.116236 Received 19 March 2020; Received in revised form 25 March 2020; Accepted 27 March 2020 Available online 09 April 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al SiO2 Non-porous SiO2 nanoparticles have been used as reinforcements for polymeric materials (Molatlhegi & Alagha, 2017) while porous SiO2 nanoparticles can be used for the allocation and release of drugs (Wu & Sailor, 2009) In the present work, non-porous SiO2 nanoparticles were used as reinforcements for the microgels and to increase the space between the chains Considering the several advantages of drug delivery systems, the present work aimed to develop efficient hybrid microgels based on chemically modified chitosan, reinforced with modified SiO2 nanoparticles, capable of completely releasing drugs in short periods It also aimed to evaluate if the microgels were pH-responsive as well as their potential application on the treatment of gastric disorders Table Factorial design for the evaluation of GMACTS (%) and SiO2-VTS (%) effect over microgels properties Sample GMA SiO2-VTS (%) MG-C1A30T4 MG-C1A30T4S MG-C2A30T4 MG-C2A30T4S MG-C2.78A30T4 MG-C2.78A30T4S 1 2 2.78 2.78 1 CTS (%) assays, the amount of vitamin-B12 utilized was correspondent to 10% of the GMACTS amount Table shows the factorial design performed to evaluate the effect of SiO2-VTS over the properties of the microgels The factors were the amount of GMACTS and SiO2-VTS The samples were named “MG-CxA30T3”, where MG means microgel, the upper letters C, A, and T means GMACTS, amplitude (equivalent to 30%) and time (equivalent to of sonication), respectively, and the sub-index ‘x’ refers to the amount, in percentage, of GMA CTS utilized Samples containing SiO2-VTS NPs also have the letter S in the name Materials and methods 2.1 Materials Mowiol poly(vinyl alcohol)® (PVA; 86.7–88.7 mol % hydrolysis, Mw ∼31.000 Da), Glycidyl methacrylate (GMA), poly(vinyl pyrrolidine K10) (PVP), chitosan (CTS; 75–85 % deacetylated, Mw 50.000–190.000 Da), tetraethylorthosilicate (TEOS), and vitamin B12 were obtained from Sigma-Aldrich Hydroquinone was obtained by Synth Vinyltrimethoxysilane (VTS) was obtained from Acros Dulbecco modified eagle medium (DMEM) and bovine fetal serum were obtained from Gibco®, and (3-(4,5-dimethyltiazol-2-il)-2-5-diphenyltetrazolium) bromide (MTT) was obtained from Amresco® All the other reactants were at an analytical degree 2.3 Characterizations 2.3.1 Fourier transform infrared (FTIR)-attenuated total reflection (ATR) The materials were characterized by FTIR-ATR (from 4000 to 400 cm−1, Perkin Elmer Equipment) to confirm the occurrence of the chemical modifications 2.2 Methods 2.2.1 Chitosan chemical modification with GMA Chitosan was modified with GMA according to the method reported by Garcia-Valdez, Champagne-Hartley, Saldivar-Guerra, Champagne, and Cunningham (2015) In brief, g of chitosan was solubilized in 100 mL of acetic acid 0.4 M previous to the addition of GMA, KOH, and hydroquinone The solution was degasified for 30 min, and then, the temperature was increased to 70 °C The system was kept under magnetic stirring and reflux for h At the end of the reaction, the solution (GMACTS) was transferred to a beaker containing 200 mL of propanone To precipitate the material, the pH was adjusted using KOH (until pH 9.0), and the final material was vacuum filtered and lyophilized (Terroni’s Scientific Equipments Enterprise Lyophilizator 2) for 24 h 2.3.2 Zeta potential Solutions at pHs ranging from 3.0 to 11.0 were prepared using a solution of NaCl mM The pH was adjusted using NaOH 0.1 M and HCl 0.1 M The samples were transferred to beakers containing a solution of specific pH value After 30 in contact with the solution, 1.5 mL of each sample was transferred for a glass cell and analyzed, in triplicate, in a Zeta Potential DLS Analyzer 2.3.3 Dynamic light scattering (DLS) The hydrodynamic diameter of the microgels was measured in a Nano Particle Size The samples were dispersed in acetone and analyzed in triplicate 2.3.4 Scanning electron microscope (SEM) For the morphology analyses, the samples were metalized for 120 s, and analyzed in a Quanta 250 SEM, operating at 15 kV acceleration voltage, and 30 mA of current intensity 2.2.2 Synthesis and modification of the SiO2 nanoparticles The SiO2 NPs were synthesized and modified according to (Simão et al., 2020) Briefly, tetraethyl orthosilicate (TEOS) was added to a solution containing water, ethanol, and NH4OH After 24 h, the solution was centrifuged, and the solid material (SiO2) was washed in a hydroalcoholic solution In the second step, the SiO2 was protected with PVP K10 (PVP-SiO2) and, then, “cut” using NaOH The final material (Cut-SiO2) was washed in a hydroalcoholic solution In the third step, the Cut-SiO2 was chemically modified by vinyl trimethoxysilane (VTS), centrifuged and lyophilized for 24 h 2.3.5 Cytotoxicity Cytotoxicity assays were performed using epithelial colorectal adenocarcinoma cells, obtained from Homo sapiens (HT-29) The cells were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium), supplemented with fetal bovine serum 10% (FBS) for 96 h, incubated at 37 °C and % CO2 tension A suspension containing 2.5 × 105 cells mL−1 was placed in a 96-wells microplate after trypsinization After 24 h of cell adhesion, different microgels concentrations (ranging from 1000 μg/mL to 50 μg/mL) were dispensed over the cells, and the microplate was incubated at the same conditions previously described The cell viability was determined after 48 h by the MTT method (3(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Amresco®) Briefly, an MTT solution was prepared at a concentration of mg mL−1 and, then, 50 μL was placed in each well The microplates were incubated during h, in the absence of light and, next, formazan crystals were solubilized in DMSO The purple color generated from the 2.2.3 Microgels synthesis The microgels were prepared through emulsion method, as described by Silva (da Silva et al., 2014) An aqueous solution (w) (based on GMACTS, PVA, and SiO2-VTS) and an organic solution (o), (based on benzyl alcohol) were sonicated for in a DP Cole Parmer Ultrasonic Processor, at 30% of amplitude, for emulsion formation Then, sodium persulfate solubilized in 200 μL of distilled water was added to the emulsion and it was sonicated for After the sonication, the microgels were precipitated in 200 mL of propanone, and washed with acetone and ethanol, three times each Then, the final material was lyophilized for 24 h For controlled release Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al mitochondrial enzymatic metabolism of viable cells was measured in a spectrophotometer microplate reader, at 570 nm 2.3.6 In vitro drug release assays For in vitro drug release assays, two pHs were evaluated: acidic (pH 1.2, adjusted with HCl) and neutral (pH 7.4, using PBS), simulating the stomach and the intestine pH, respectively In a beaker, mg of microgel was put in direct contact with 15 mL of solution, incubated in a refrigerated shaker (Nova Tecnica - Laboratory Equipments), at 37 °C, and stirred at 50 RPM Samples were collected at specific time intervals, centrifuged for 30 s previous to the UV/Vis analysis, and analyzed in a UV/Vis spectrophotometer (Thermo Scientific Genesys 10S) After the analysis, the samples were returned to the beaker Vitamin B12 absorbance was measured at 360 nm (Sitta et al., 2014) The release mechanism was evaluated by the models of Weibull (Eq (1)) (Dash, Murthy, Nath, & Chowdhury, 2010) and allometric (Eq (2)) (Ritgers-Peppas) (Ritger & Peppas, 1987) In Eq (1), k is a release rate constant (min−1), which is characteristic of the microgel, a is a timedependent scale parameter, n is the diffusion coefficient, Ctime and Cequilibrium refers to the concentration at a specific time (x) and the equilibrium, respectively; xc refers to the time-lag (time previous to the start of the release, xc = in the present work) n Ctime = − e−(k (x − x 0) ) ; k = Cequilibrium a (1) −1 In Eq (2), k is a release rate constant (min ) and n, the diffusion coefficient The allometric model only considers 60 % of the release Ctime = kx n Cequilibrium Fig (a) Reaction schema of the chemical modification of chitosan (CTS) by glycidyl methacrylate (GMA), and (b) FTIR-ATR spectra for CTS and GMACTS (2) analysis (Fig 2(a)) The chemical modification of the cut-SiO2 NPs was confirmed by FTIR analysis (Fig 2(a)) It was confirmed by a vinyl stretching band, observed at 1560 cm−1 Also, the angular deformation of C–H was observed at 1440 cm−1 (Liu et al., 2017) Still, the asymmetric stretching of Si-O-Si was observed at 1070 cm−1 (Liu et al., 2017) The hydrodynamic diameter of the SiO2-VTS NPs was evaluated by TEM (Fig 2(b)), and DLS analysis (Fig 2(c)) The TEM results indicated a particle size of (194.4 ± 17.6) nm The DLS results indicated that the diameter of most particles (36%) was 190 nm However, the number of particles at 160 nm was 35% It explains the standard deviation observed at the TEM analysis Since both results (TEM and DLS) are in accordance, it is confirmed that the synthesis led to particles at two main diameter sizes Similar results were found by Nozawa et al (2005), whose diameter was 200 nm for SiO2 NPs, following the method of Stöber Nevertheless, Simão et al (2020) observed diameters of 150 nm for SiO2 NPs also modified by VTS Results and discussion 3.1 Chemical modification of CTS by GMA (GMACTS) The chemical modification reactions were performed to add vinyl groups to the CTS chains The reaction occurred in an acidic medium, promoting the opening of the epoxy ring from GMA (Reis et al., 2008) A schema of the reaction indicating the two possible products is shown in Fig 1(a), considering the reaction in polar protic conditions (Reis et al., 2008) The chemical modification of CTS by GMA was confirmed by the band at 1550 cm−1 (Fig 1(b)) This band can be attributed to the axial deformation of the C]C from GMA (Reis et al., 2008) Other chitosan characteristic bands can also be observed in Fig 1(b) For example, the broadband ranging from 3650 to 3200 cm−1, can be attributed to the stretching of NH2 and OH from CTS (de Souza Costa & Mansur, 2008) while the band at 1650 can be attributed to the stretching of C]O (Reis et al., 2008) This carbonyl group is observed in the acetylated portion of chitosan CTS 3.3 Characterization of the GMA CTS microgels 3.3.1 Morphology Fig shows the SEM results A non-homogeneity of particles was observed in all of the obtained samples However, all chitosan microgels showed a rough and non-porous surface The microgels without SiO2-VTS were more spherical than the ones containing the NPs It might have happened due to a more organized and compact arrangement of chains On the other hand, the microgels containing SiO2-VTS showed irregular shapes, and particle aggregation was observed for samples MGC1A30T3S and MG-C2A30T3S The intense aggregation observed for sample MG-C1A30T3S might have happened due to the combination of the synthetic method, the polydispersity of chitosan, and the uncontrolled polymerization reaction Even though the emulsion was formed before the addition of the radical initiator, reactors (drops of 3.2 Synthesis and characterization of SiO2-VTS The method of Stöber (Stöber, Fink, & Bohn, 1968) has been widely used for the synthesis of SiO2 NPs (Wong et al., 2011) It is known that the reaction mechanism is based on the hydrolysis of TEOS followed by a condensation step (Nozawa et al., 2005; Van Blaaderen, Van Geest, & Vrij, 1992) In the second step, the SiO2 NPs were protected with PVP to avoid the aggregation of the particles It eases the nucleophilic attack promoted by NaOH Furthermore, this attack is responsible to promote the ‘cutting’ of SiO2 chains It was also expected to increase the surface area, which is crucial for the graftization promoted in the following step (Simão et al., 2020) Since the second does not involve any chemical modifications, only SiO2 characteristic bands were observed at the FTIR Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al Fig (a) FTIR spectra of SiO2, cut-SiO2, and SiO2-VTS; (b) TEM of the SiO2-VTS nanoparticles, and (c) DLS analysis of the SiO2-VTS nanoparticles 3.3.2 Zeta potential Zeta potential influences directly the stability of suspensions, the interaction between charged drugs and polymeric microspheres, and the adhesion of devices on biologic interfaces (Berthold, Cremer, & Kreuter, 1996) In the present work, from pH to 9, all samples showed positive charges on their surfaces (Fig 4) Among the positive zeta potential values, the highest one (28 mV) was observed for sample MG-C1A30T3 (Fig 4(a)) while the lowest one (13.9 mV), for sample MG-C2.78A30T3 (Fig 4(e)) The positive values were expected in acidic pH values because the modified chitosan has polar groups (NH, C]O, and OH) in its structure In acidic pHs, these groups are positively protonated due to the excess of H+ in the medium Considering that the pKa of chitosan is 6.3, no charges were supposed to be observed at pH However, until pKa = 6.9, about 20% of the amino-groups are still expected to be protonated (Muzzarelli, 1977) It explains the observed positive charges Nevertheless, at pH 7, the zeta potential values observed in this work ((18.7 ± 2.4) mV) are considerably higher than the ones observed by Tourrette et al (2009) In their work, they synthesized microgels based on poly(isopropylacrylamide) and chitosan At pH 7, the observed zeta potential was approximately 1.8 mV The zeta potential values at pH also explain the aggregation observed at the SEM analysis (Fig 3) More stable particles have high zeta potential values (Hunter, Ottewill, & Rowell, 2013) because their repulsive forces are strong enough to prevent particle aggregation In basic pHs, the zeta potential was supposed to be negative because the polar groups from chitosan are deprotonated Among the negative modified chitosan dispersed in the oil phase) of different sizes were formed The sample MG-C2A30T3S might have been formed by modified chitosan oligomers It led to the smaller reactors and, consequently, smaller microgels It is also important to highlight that PVA was added to act as a surfactant in the medium, preventing the aggregation of particles (Zeng et al., 2004) However, it was not efficient enough in all of the samples Also, the SiO2-VTS NPs were supposed to act as both “spacers” and reinforcements But the presence of negative charges in the reactors might have affected the stability of the microgels Since chitosan has polar groups, an attraction between the protonated amino-groups and the negative charges in the surface of the SiO2-VTS NPs (Panão et al., 2019) might have occurred It would have affected the organization and distribution of the polymeric chains and the nanoparticles, favoring coalescence and aggregation Another important factor to be considered is the amount of CTS used in each synthesis Higher amounts of chitosan also increased the number of amino-groups in the medium It affected the net charge in the surface of the microgels, as shown by the zeta potential analysis (Section (3.3.2)) Regarding the diameter of the microgels, the mean value obtained for the gels without SiO2-VTS was (18.7 ± 12.3) μm while it was (11.3 ± 8.07) μm for the gels containing SiO2-VTS The high standard deviation values are explained by the several problems in the synthesis (non-uniformity of reactors, chitosan high polydispersity, and a noncontrolled polymerization reaction) Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al Fig SEM images from (a) MG-C1A30T3; (b) MG-C1A30T3S; (c) MG-C2A30T3; (d) MG-C2A30T3S; (e) MG-C2.78A30T3; (f) MG-C2.78A30T3S obtained for the model of Weibull because the correlation coefficient (R²) values were higher for this model The values obtained for both models are present in Table At pH 7.4, a Fickian release (Rdif < < Rrelax) (Masaro & Zhu, 1999) was observed for all the samples In this mechanism, the solvent diffusion rate (Rdif) is smaller than the polymeric relaxation rate (Rrelax), (Rdif < < Rrelax) (Masaro & Zhu, 1999) All the other samples reached equilibrium before 30 This fast release might have happened due to the high hydrophilicity of vitamin-B12, preferring the release medium instead of the sample Also, repulsions between the protonated amino-groups might have affect the arrangement of the chains, allowing them to expand This expansion eases the scape of vitamin-B12 At pH 1.2, the samples MG-C1A30T3, MG-C1A30T3S e MG-C2A30T3 reached the equilibrium after about 20 min, while for samples MGC2A30T3S, MG-C2.78A30T3 and MG-C2.78A30T3S, it happened after 40 For all the samples, but MG-C1A30T3S, whose release is complex, the observed mechanism was Fickian (Rdif < < Rrelax) (Masaro & Zhu, 1999) The release rate constant (k) at pH 1.2, indicated a fast release for the sample MG-C1A30T3S (3.23 ± 2.71) min−1 and a slower one for the sample MG-C2A30T3 S (0.12 ± 0.01) min−1 Even though the equilibrium was reached after a short time, a more controlled release was observed at pH 1.2 Thus, it is concluded that the material was more responsive in acidic pHs Considering the repulsive forces caused by the positive charges in the microgels and the positive charges from the release medium, the expansion observed in the chains might have been smaller The expansion occurs until a state of higher stability is reached However, in the excess of repulsive forces, this values, the lowest zeta potential (-5.62 mV) was observed for sample MG-C1A30T3 (Fig (a)) However, positive zeta potentials were observed at pH 11 for the samples MG-C2A30T3 (Fig (b)) and MGC2.78A30T3S (Fig (f)) It could have happened due to non-neutralized amino groups (Berthold et al., 1996) 3.3.3 Cytotoxicity MTT is a quick and versatile colorimetric method where cells show the ability to reduce MTT, indicating mitochondrial activity and integrity (cell integrity) (Mao et al., 2004) The obtained results (Fig 5) confirmed that the microgels are not toxic for HT-29 cells, once cell viability was almost 100% even for the highest concentrations of microgels (1000 μg mL−1) The high cytocompatibility was expected because chitosan is a biocompatible polymer Although Yang et al (2016) observed a considerable decrease in cell survival (∼ 50%) at high concentrations of SiO2 NPs (750 μg mL−1), in low concentrations, they not affect the cytocompatibility It was observed by Simão et al (2020), whose hydrogels based on chondroitin sulfate, casein, and SiO2 led to cytocompatibility values higher than 80% Therefore, the amount of SiO2 used in the present work did not offer risks to cell viability, confirming the potential application of these microgels in biological environments 3.3.4 Controlled release assays in vitro assays of controlled release gives information about the releasing mechanism of each matrix in simulated physiological environments (Dengre, Bajpai, & Bajpai, 2000) Fig shows the results Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al Fig Zeta potential of the samples: (a) MG-C1A30T3; (b) MG-C1A30T3S; (c) MG-C2A30T3; (d) MG-C2A30T3S; (e) MG-C2.78A30T3; (f) MG-C2.78A30T3S treatment of gastric wounds, like ulcers (Patel & Amiji, 1996) because their release is sustained for one hour Depending on the kind of ingested food, the digestion will last about h (Malagelada, Longstreth, Summerskill, & Go, 1976) This way, devices with long-term releases are not too interesting because their activity time is limited and they would be eliminated before releasing all the entrapped drugs Similar release results were found by Kang and Kim (2010) They synthesized chitosan microgels covered with poly(N-isopropylacrylamide-co-methacrylic acid) (P(NIPAM-co-MAA)) For all the evaluated conditions, the equilibrium was reached after h They also evaluated the temperature effect over the release profile In acidic pHs, the covered microgels showed higher releases It could have happened due to co-polymer thermal contraction, creating a condensed layer that, consequently, suppressed the release Microgels degradation The pH effect over the microgels is presented in Fig It is known that the burst release can compromise the structure of the device, and decrease its lifetime, and performance (Patel & Amiji, 1996) Significant degradation signs were observed in both pHs However, it was more intense at pH 1.2 The high acidity of the medium weakens the covalent bond responsible for sustaining the structure of the microgel (Zhang, Mardyani, Chan, & Kumacheva, 2006) It might have compromised the efficiency of the drug release because the structure ruptures increased the surface area, allowing the release of higher amounts of the drug Fewer damages were observed at pH 7.4 But the structure was also compromised The damages were more significant for samples MGC2A30T3 and MG-C2A30T3S Large pores could be observed on their Fig In vitro cytotoxicity of the chitosan microgels expansion is limited Another interesting behavior observed in the present work is the initial fast release It is called burst release and it happens before a stable release profile is reached (Huang & Brazel, 2001) The adsorption of drugs on the surface of the microgels and the high solubility of vitamin-B12 in polar environments (Moreno & Salvado, 2000) might have contributed to the observed burst release (Dengre et al., 2000) The burst release is interesting for wound treatment because it promotes an immediate relief of the symptoms If followed by a slower release, it allows a gradual recovery of the damaged area (Huang & Brazel, 2001) This way, these microgels can be very useful for the Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al Fig Controlled release of vitamin-B12 at pH 1.2 and pH 7.4: (a) MG-C1A30T3; (b) MG-C1A30T3S; (c) MG-C2A30T3; (d) MG-C2A30T3S; (e) MG-C2.78A30T3; (f) MGC2.78A30T3S Table Weibull’s and allometric’s parameters for vitamin-B12 controlled release at pH 1.2 and pH 7.4: release rate constant (k) and diffusion coefficient (n) Model of Weibull Allometric model Sample pH K (min−1) n MG-C1A30T3 1.2 7.4 1.2 7.4 1.2 7.4 1.2 7.4 1.2 7.4 1.2 7.4 0.51 0.14 3.23 0.43 0.37 3.40 0.12 0.79 0.14 5.53 0.48 0.17 0.44 2.50 0.26 0.36 0.52 0.19 0.67 0.39 0.66 0.22 0.52 0.54 MG-C1A30T3S MG-C2A30T3 MG-C2A30T3S MG-C2.78A30T3 MG-C2.78A30T3S ± ± ± ± ± ± ± ± ± ± ± ± 0.15 0.01 2.71 0.28 0.01 2.09 0.01 0.20 0.01 5.45 0.02 0.01 ± ± ± ± ± ± ± ± ± ± ± ± 0.07 0.39 0.06 0.12 0.01 0.03 0.04 0.04 0.03 0.04 0.03 0.05 R² Release mechanism K (min 0.99 0.98 0.99 0.95 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 Fickian Complex Fickian 0.81 0.83 0.81 0.57 0.80 0.68 0.39 0.75 0.40 0.81 0.66 0.48 ± ± ± ± ± ± ± ± ± ± ± ± −1 ) 0.01 0.04 0.06 0.06 0.04 0.04 0.04 0.02 0.03 0.03 0.03 0.02 n 0.05 0.04 0.05 0.14 0.04 0.10 0.23 0.07 0.23 0.05 0.11 0.18 ± ± ± ± ± ± ± ± ± ± ± ± 0.01 0.01 0.02 0.03 0.01 0.02 0.03 0.01 0.03 0.01 0.01 0.01 R² Release mechanism 0.94 0.63 0.46 0.85 0.60 0.82 0.91 0.89 0.97 0.76 0.85 0.97 Pseudo-Fickian *Samples were named “MG-CxA30T3”, where MG means microgel, the upper letters C, A, and T means GMACTS, amplitude (30%) and time (3 min), respectively, and the sub-index ‘x’ refers to the amount, in percentage, of GMACTS utilized Samples containing SiO2-VTS NPs also have the letter S in the name Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al Fig SEM images after controlled release assays at pH 1.2 and pH 7.4 for (a) MG-C1A30T3; (b) MG-C1A30T3S; (c) MG-C2A30T3; (d) MG-C2A30T3S; (e) MG-C2.78A30T3; (f) MG-C2.78A30T3S Carbohydrate Polymers 239 (2020) 116236 M.C Galdioli Pellá, et al surfaces after the release assay It might have happened due to the larger expansion of the chains, as discussed in Section 3.3.4 It is also possible to conclude that the SiO2 NPs were not efficient enough in reinforcing the structure of the microgels No similar degradation results were found in the literature However, Wang, Lin, Nune, and Misra (2016) synthesized microgels based on chitosan, gelatin, N-hydroxysuccinimide (NHS) and poly (ethylineglycol) for controlled release They observed gelatin degradation after days of analysis Nevertheless, no significant structural alterations were observed da Silva, E P., Sitta, D L A., Fragal, V H., Cellet, T S P., Mauricio, M R., Garcia, F P., Kunita, M H (2014) Covalent TiO 2/pectin microspheres with Fe O nanoparticles for magnetic field-modulated drug delivery International Journal of Biological Macromolecules, 67, 43–52 Dash, S., Murthy, P N., Nath, L., & Chowdhury, P (2010) Kinetic modeling on drug release from controlled drug delivery systems Acta Poloniae Pharmaceutica, 67(3), 217–223 de Souza Costa, E., Jr., & Mansur, H S (2008) Preparaỗóo e caracterizaỗóo de blendas de quitosana/poli (ỏlcool vinớlico) reticuladas quimicamente com glutaraldeớdo para aplicaỗóo em engenharia de tecido Quimica Nova, 31(6), 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Silva, E P., Valente, A J M., Muniz, E C., & Rubira, A F (2014) Drug release mechanisms of chemically cross-linked albumin Conclusion Hybrid microgels based on modified chitosan and SiO2-VTS NPs were synthesized by emulsion polymerization The hydrodynamic diameter of the microgels ranged between (18.7 ± 12.3) μm and (11.3 ± 8.07) μm for the gels without and with SiO2-VTS, respectively The SiO2-VTS NPs were added to act “spacers” and reinforcements to the structure of the microgels Regarding the drug release behavior, a burst release of vitamin-B12 was observed for all the samples, and the equilibrium was reached before h for all samples The main observed release mechanism was Fickian, which is characterized by a drug diffusion rate smaller than the relaxation rate At pH 7.4, only sample MGC1A30T3 showed a complex release Despite the burst release, a more controlled release was accomplished in acidic medium (pH 1.2) Severe degradation was observed in all of the microgels, especially at pH 1.2, suggesting a weakening of the chemical bonds responsible for sustaining the structure of the microgel It also suggests that the SiO2-VTS NPs were not efficient reinforcements Therefore, the properties observed for these microgels are interesting for gastric wound treatments because they are capable of promoting a fast release, which controls the pain The followed slower release sustains the effect of the drug and improves the efficiency of the treatment This fast release also ensures that all the loaded drug will have been completely released before the device leaves the stomach CRediT authorship contribution statement Michelly Cristina Galdioli Pellá: Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization Andressa Renatta Simão: Writing - review & editing, Visualization Michele Karoline Lima-Tenório: Conceptualization Ernandes Tenório-Neto: Conceptualization Débora Botura Scariot: Formal analysis Celso Vataru 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