Chitosan hybrid microgels for oral drug delivery

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.

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Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol

Chitosan hybrid microgels for oral drug delivery

Michelly Cristina Galdioli Pelláa,* , Andressa Renatta Simãoa, Michele Karoline Lima-Tenóriob,

Ernandes Tenório-Netob, Débora Botura Scariotc, Celso Vataru Nakamurac, Adley Forti Rubiraa,*

a Department of Chemistry, State University of Maringa, Av Colombo, 5790, CEP, 87020-900, Maringa, Parana, Brazil

b Department of Chemistry, State University of Ponta Grossa, Av Gen Carlos Cavalcanti, 4748, CEP 84030-900, Ponta Grossa, Parana, Brazil

c Department of Basic Science of Health, State University of Maringa, Av Colombo, 5790, CEP 87020-900, Maringa, Parana, Brazil

A R T I C L E I N F O

Keywords:

Chemical modification

SiO 2 nanoparticles

Glycidyl methacrylate

A B S T R A C T

In the present work, hybrid microgels based on chitosan and SiO2nanoparticles (NPs) were synthesized Both chitosan and the SiO2NPs were submitted to chemical modification reactions to having vinyl groups in-corporated 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-respon-siveness, especially in acidic pHs The longest release lasted 45 min 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

1 Introduction

The development of devices with efficient controlled release

beha-vior 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

admini-strated (Park, 1988), implanted (Cohn, Sosnik, & Garty, 2005) or

in-jected 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,

electro-magnetic field (Grainger, 2013), etc They also can swell water,

ex-panding 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 po-sitively charged polymer whose chains can be easily modified (Zeng, Fang, & Xu, 2004) This polymer is biocompatible, biodegradable, non-toxic (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 na-noparticles (Grainger, 2013;Lu, Zahedi, Forman, & Allen, 2014) These inorganic nanoparticles can be biocompatible, non-toxic, and bioab-sorpt (Soares et al., 2016) They have been being combined with polymeric materials like, for example, aiming to improve the drug de-livery system (Lu et al., 2014)

One example of inorganic nanoparticle with attractive properties is

https://doi.org/10.1016/j.carbpol.2020.116236

Received 19 March 2020; Received in revised form 25 March 2020; Accepted 27 March 2020

⁎Corresponding authors

E-mail addresses:michellepella57@gmail.com(M.C Galdioli Pellá),afrubira@gmail.com(A.F Rubira)

Available online 09 April 2020

0144-8617/ © 2020 Elsevier Ltd All rights reserved

T

SiO2 Non-porous SiO2nanoparticles 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 SiO2nanoparticles were

used as reinforcements for the microgels and to increase the space

be-tween 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

nano-particles, 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

2 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

2.2 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, 1 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

mag-netic stirring and reflux for 2 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 thefinal material

was vacuumfiltered and lyophilized (Terroni’s Scientific Equipments

Enterprise Lyophilizator 2) for 24 h

2.2.2 Synthesis and modification of the SiO2nanoparticles

The SiO2NPs 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

hy-droalcoholic solution In the second step, the SiO2was 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-SiO2was chemically modified by vinyl trimethoxysilane (VTS),

centrifuged and lyophilized for 24 h

2.2.3 Microgels synthesis

The microgels were prepared through emulsion method, as

de-scribed by Silva (da Silva et al., 2014) An aqueous solution (w) (based

onGMACTS, PVA, and SiO2-VTS) and an organic solution (o), (based on

benzyl alcohol) were sonicated for 3 min in a DP Cole Parmer

Ultra-sonic Processor, at 30% of amplitude, for emulsion formation Then,

sodium persulfate solubilized in 200μL of distilled water was added to

the emulsion and it was sonicated for 1 min

After the sonication, the microgels were precipitated in 200 mL of

propanone, and washed with acetone and ethanol, three times each

Then, thefinal material was lyophilized for 24 h For controlled release

assays, the amount of vitamin-B12 utilized was correspondent to 10%

of theGMACTS amount

Table 1shows the factorial design performed to evaluate the effect

of SiO2-VTS over the properties of the microgels The factors were the amount ofGMACTS and SiO2-VTS

The samples were named “MG-CxA30T3”, where MG means mi-crogel, the upper letters C, A, and T means GMACTS, amplitude (equivalent to 30%) and time (equivalent to 3 min of sonication), re-spectively, 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

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

2.3.2 Zeta potential Solutions at pHs ranging from 3.0 to 11.0 were prepared using a solution of NaCl 1 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 min 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

2.3.5 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 5 % CO2tension A suspension containing 2.5 × 105cells

mL−1was placed in a 96-wells microplate after trypsinization After 24 h of cell adhesion, different microgels concentrations (ranging from 1000μg/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

2 mg mL−1and, then, 50μL was placed in each well The microplates were incubated during 4 h, in the absence of light and, next, formazan crystals were solubilized in DMSO The purple color generated from the

Table 1 Factorial design for the evaluation ofGMACTS (%) and SiO2-VTS (%) effect over microgels properties

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, 5 mg of

mi-crogel 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

ab-sorbance 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

time-dependent scale parameter, n is the diffusion coefficient, Ctime and

Cequilibrium refers to the concentration at a specific time (x) and the

equilibrium, respectively; xcrefers to the time-lag (time previous to the

start of the release, xc= 0 in the present work)

C

1

time

equilibrium

k x x

( ( ) )n

(1)

In Eq.(2), k is a release rate constant (min−1) and n, the diffusion

coefficient The allometric model only considers 60 % of the release

=

C

time

equilibrium

n

(2)

3 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 inFig 1(b) For example, the

broadband ranging from 3650 to 3200 cm−1, can be attributed to the

stretching of NH2and 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

3.2 Synthesis and characterization of SiO2-VTS

The method of Stöber (Stöber, Fink, & Bohn, 1968) has been widely

used for the synthesis of SiO2NPs (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 SiO2NPs were protected with PVP to avoid

the aggregation of the particles It eases the nucleophilic attack

pro-moted by NaOH Furthermore, this attack is responsible to promote the

‘cutting’ of SiO2chains 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 SiO2characteristic bands were observed at the FTIR

analysis (Fig 2(a))

The chemical modification of the cut-SiO2NPs 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 ob-served 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 SiO2NPs also modified by VTS

3.3 Characterization of theGMACTS microgels

3.3.1 Morphology Fig 3shows the SEM results A non-homogeneity of particles was observed in all of the obtained samples However, all chitosan micro-gels showed a rough and non-porous surface The micromicro-gels without SiO2-VTS were more spherical than the ones containing the NPs It might have happened due to a more organized and compact arrange-ment of chains

On the other hand, the microgels containing SiO2-VTS showed ir-regular shapes, and particle aggregation was observed for samples

MG-C1A30T3S 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 un-controlled polymerization reaction Even though the emulsion was formed before the addition of the radical initiator, reactors (drops of

Fig 1 (a) Reaction schema of the chemical modification of chitosan (CTS) by glycidyl methacrylate (GMA), and (b) FTIR-ATR spectra for CTS andGMACTS

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

(uniformity of reactors, chitosan high polydispersity, and a

non-controlled polymerization reaction)

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 3 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 be-cause 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 sup-posed to be observed at pH 7 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 byTourrette et al (2009)

In their work, they synthesized microgels based on poly(iso-propylacrylamide) and chitosan At pH 7, the observed zeta potential was approximately 1.8 mV

The zeta potential values at pH 7 also explain the aggregation ob-served at the SEM analysis (Fig 3) More stable particles have high zeta potential values (Hunter, Ottewill, & Rowell, 2013) because their re-pulsive 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

Fig 2 (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

values, the lowest zeta potential (-5.62 mV) was observed for sample

MG-C1A30T3(Fig 4(a)) However, positive zeta potentials were

ob-served at pH 11 for the samples MG-C2A30T3 (Fig 4 (b)) and

MG-C2.78A30T3S (Fig 4(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

in-tegrity (cell inin-tegrity) (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

mi-crogels (1000μg mL−1)

The high cytocompatibility was expected because chitosan is a

biocompatible polymer AlthoughYang et al (2016)observed a

con-siderable decrease in cell survival (∼ 50%) at high concentrations of

SiO2NPs (750μg mL−1), in low concentrations, they do not affect the

cytocompatibility It was observed bySimão et al (2020), whose

hy-drogels based on chondroitin sulfate, casein, and SiO2 led to

cyto-compatibility 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

re-leasing mechanism of each matrix in simulated physiological

environ-ments (Dengre, Bajpai, & Bajpai, 2000) Fig 6 shows the results

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 inTable 2

At pH 7.4, a Fickian release (Rdif< < Rrelax) (Masaro & Zhu, 1999) was observed for all the samples In this mechanism, the solvent dif-fusion rate (Rdif) is smaller than the polymeric relaxation rate (Rrelax), (Rdif< < Rrelax) (Masaro & Zhu, 1999) All the other samples reached equilibrium before 30 min 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, al-lowing 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

MG-C2A30T3S, MG-C2.78A30T3 and MG-C2.78A30T3S, it happened after

40 min For all the samples, but MG-C1A30T3S, whose release is com-plex, 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−1and a slower one for the sample MG-C2A30T3S (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

Fig 3 SEM images from (a) MG-C1A30T3; (b) MG-C1A30T3S; (c) MG-C2A30T3; (d) MG-C2A30T3S; (e) MG-C2.78A30T3; (f) MG-C2.78A30T3S

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

vi-tamin-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

treatment of gastric wounds, like ulcers (Patel & Amiji, 1996) because their release is sustained for one hour Depending on the kind of in-gested food, the digestion will last about 2 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 byKang and Kim (2010) They synthesized chitosan microgels covered with poly(N-iso-propylacrylamide-co-methacrylic acid) (P(NIPAM-co-MAA)) For all the evaluated conditions, the equilibrium was reached after 1 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 inFig 7 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 mi-crogel (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

MG-C2A30T3 and MG-C2A30T3S Large pores could be observed on their

Fig 4 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

Fig 5 In vitro cytotoxicity of the chitosan microgels

Fig 6 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)

MG-C2.78A30T3S

Table 2

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)

MG-C 1 A 30 T 3 1.2 0.51 ± 0.15 0.44 ± 0.07 0.99 Fickian 0.81 ± 0.01 0.05 ± 0.01 0.94 Pseudo-Fickian

7.4 0.14 ± 0.01 2.50 ± 0.39 0.98 Complex 0.83 ± 0.04 0.04 ± 0.01 0.63 MG-C 1 A 30 T 3 S 1.2 3.23 ± 2.71 0.26 ± 0.06 0.99 Fickian 0.81 ± 0.06 0.05 ± 0.02 0.46

MG-C 2 A 30 T 3 S 1.2 0.12 ± 0.01 0.67 ± 0.04 0.99 0.39 ± 0.04 0.23 ± 0.03 0.91

MG-C 2.78 A 30 T 3 1.2 0.14 ± 0.01 0.66 ± 0.03 0.99 0.40 ± 0.03 0.23 ± 0.03 0.97

MG-C 2.78 A 30 T 3 S 1.2 0.48 ± 0.02 0.52 ± 0.03 0.99 0.66 ± 0.03 0.11 ± 0.01 0.85

*Samples were named“MG-CxA30T3”, where MG means microgel, the upper letters C, A, and T meansGMACTS, amplitude (30%) and time (3 min), respectively, and the sub-index‘x’ refers to the amount, in percentage, ofGMACTS utilized Samples containing SiO2-VTS NPs also have the letter S in the name

Fig 7 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

surfaces after the release assay It might have happened due to the

larger expansion of the chains, as discussed in Section3.3.4 It is also

possible to conclude that the SiO2NPs 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

degrada-tion after 7 days of analysis Nevertheless, no significant structural

al-terations were observed

4 Conclusion

Hybrid microgels based on modified chitosan and SiO2-VTS NPs

were synthesized by emulsion polymerization The hydrodynamic

dia-meter 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 1 h for all samples The main observed

release mechanism was Fickian, which is characterized by a drug

dif-fusion rate smaller than the relaxation rate At pH 7.4, only sample

MG-C1A30T3showed 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

sus-taining the structure of the microgel It also suggests that the SiO2-VTS

NPs were not efficient reinforcements Therefore, the properties

ob-served 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 Nakamura: Supervision Adley Forti

Rubira: Supervision

Acknowledges

The authors are grateful to the Coordenação de Aperfeiçoamento de

Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq) for the financial support M K

Lima-Tenório thanks the Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq) - Brasil for post-doctorate fellowship (process N°

150268/2016-5)

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