Magnetic microspheres based on pectin coated by chitosan towards smart drug release

This study reports the preparation of microspheres of pectin and magnetite nanoparticles coated by chitosan to encapsulate and deliver drugs. Magnetic-pectin microspheres were obtained by ionotropic gelation followed by polyelectrolyte complexation with chitosan.

Available online 2 April 2021

0144-8617/© 2021 Elsevier Ltd This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/)

Magnetic microspheres based on pectin coated by chitosan towards smart

drug release

Thalia S.A Lemos , Jaqueline F de Souza , Andr´e R Fajardo *

Laborat´orio de Tecnologia e Desenvolvimento de Comp´ositos e Materiais Polim´ericos (LaCoPol), Universidade Federal de Pelotas (UFPel), Campus Cap˜ao do Le˜ao s/n,

96010-900, Pelotas, RS, Brazil

A R T I C L E I N F O

Keywords:

Magnetic

Biopolymers

Microspheres

Smart materials

Stimuli-responsive system

Drug delivery

A B S T R A C T This study reports the preparation of microspheres of pectin and magnetite nanoparticles coated by chitosan to encapsulate and deliver drugs Magnetic-pectin microspheres were obtained by ionotropic gelation followed by polyelectrolyte complexation with chitosan Characterization data show that magnetite changes the physico-chemical and morphological properties of the microspheres compared to the non-magnetic samples Using metamizole (Mtz) as a drug model, the magnetic microspheres showed appreciable encapsulation efficiency (85

%) Release experiments performed in simulated gastric (pH 1.2) and intestinal (pH 6.8) fluids suggested that the release process is pH-dependent At pH 6.8, the Mtz release is favored achieving 75 % after 12 h The application

of an external magnetic field increased the release to 91 % at pH 6.8, indicating that the release also is magnetic- dependent The results suggest that the magnetic microspheres based on pectin/chitosan biopolymers show the potential to be used as a multi-responsive drug delivery system

1 Introduction

The first examples of drug delivery systems (DDS) based on polymers

were reported almost five decades ago and have since attracted the

attention of several researcher fields (Wong et al., 2018) In summary,

this success is attributable to the many advantages offered by these

delivery systems as compared to free-drug formulations Some attributes

of polymeric DDS include the ability to maintain drug concentration

within a desirable range, increase drug bioavailability, a decrease of side

effects and administration doses, and increase of patient compliance to

the treatment (Gunter et al., 2018; Wong et al., 2018) Overall, these

features allowed enhancing the efficiency of several drugs and

medica-ment treatmedica-ments for various diseases and conditions (Jafari et al., 2020;

Li et al., 2020)

Nowadays, the main challenges related to the development of more

efficient polymeric DDS are related to the improvement of drug

encap-sulation efficiency and release (Patra et al., 2018) Specifically, drug

release is a critical stage since it is related to the success of the DDS The

release of a drug (or other bioactive compounds) from a polymeric

system can occur continuously or cyclically over a long period or it can

be triggered by an external stimulus (Karimi et al., 2016) This last

mechanism has gained importance as an efficient strategy to overcome

two potential shortcomings related to the releasing process: (i) the inability to deliver the loaded drug and (ii) burst release effects (Pham

et al., 2020) In recent years, researchers have developed polymeric DDS able to control their release mechanism according to changes on different environmental parameters (such as pH condition, temperature, ionic strength, light incidence, and electric and magnetic fields) (Raza

et al., 2019; Thevenot et al., 2013) Although these parameters can be modulated under the physiological environment, in which the DDS is administrated, some of them can be invasive and cause undesired effects (Senapati et al., 2018) In light of this, some authors claim that the use of DDS endowed with stimuli-responsive magnetic properties is a prom-ising alternative to overcome the aforementioned limitations (Frachini

& Petri, 2019; Price et al., 2018) The efficiency of these responsive systems can be ascribed to the use of external magnetic fields, which enable controlling the DDS actuation remotely According to Farah (2016), the main advantage of magnetic-responsive DDS is the reduction

in the dose and side effects of the drug Additionally, therapeutic re-sponses in target organs can be achieved by a small fraction of the free drug due to the improvement of the drug bioavailability The magnetic response is typically obtained by focusing an extracorporeal magnetic, which is less invasive than other responsive systems (Mura et al., 2013) Iron oxides such as Fe3O4 (magnetite) and γ-Fe2O3 (maghemite) have

* Corresponding author

E-mail address: andre.fajardo@pq.cnpq.br (A.R Fajardo)

Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2021.118013

Received 29 January 2021; Received in revised form 27 February 2021; Accepted 26 March 2021

been predominantly used to induce magnetic properties in polymeric

DDS because of their biocompatibility and low toxicity properties

(Ghazanfari et al., 2016) Moreover, the affinity of these oxides with

water allows the interaction of the same with different biological

spe-cies Consequently, the incorporation of these oxides into natural

ma-terials like polysaccharides may result in smart drug delivery systems

The use of polysaccharides is preferred by several investigators devoted

to preparing magnetic DDS since they enable a good dispersion and

stabilization of the iron oxide particles (Chang et al., 2011) Of course,

the use of polysaccharides in the preparation of DDS is also stimulated

owing to their interesting properties such as biocompatibility,

biode-gradability, non-toxicity, renewability, low-cost, and processability (Oh

et al., 2009) Among the polysaccharides suitable to this application,

pectin, a natural polymer component of all plant cell walls has been

poorly explored Pectin (Pec) is a complex polysaccharide,

predomi-nantly linear, consisting mainly of methoxy esterified α(1→4)-linked

D-galacturonic acid units that according to their esterification degree

can form gels (Lara-Espinoza et al., 2018) Capel et al (2006)

demon-strate that Pec with a low esterification degree undergoes ionotropic

crosslinking in the presence of Ca2+ions resulting in a stable hydrogel

This gel-forming ability of Pec can also be useful to form polyelectrolyte

complexes with polycationic species, like chitosan, a well-known chitin

derivative Chitosan (Cs), a linear copolymer polysaccharide consisting

of β(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine units

widely used in pharmaceutical and biomedical applications owing to its

biological properties (Younes & Rinaudo, 2015) The protonable amino

groups of Cs can interact strongly with the carboxylate-rich structure of

Pec resulting in a polyelectrolyte complex (Rampino et al., 2016)

Earlier studies demonstrated that the stability of Pec/Cs complexes can

be modified by changing external conditions like pH and temperature,

which allows ranking these materials as potential DDS with sensitive

properties (Maciel et al., 2015; Sigaeva et al., 2020)

Herein, we prepared microspheres consisting of pectin and

magne-tite nanoparticles, which were coated by a chitosan layer, and

hypoth-esize that they can be used as a multi-responsive DDS The magnetic

microspheres were loaded with metamizole (Mtz), which is a pyrazolone

derivative commonly used to treat various pain conditions (e.g.,

post-operative pain, colic pain, cancer pain, and migraine) in humans and

veterinary practices (Jasiecka et al., 2014) A series of experiments were

performed to investigate the behavior and mechanism associated with

the Mtz release under different simulated physiological conditions

(gastric and intestinal fluids) and with and without the presence of an

external magnetic field

2 Materials and methods

2.1 Materials

Orange (Citrus sinensis) peels were obtained from the student

restaurant at Universidade Federal de Pelotas (Pelotas, RS, Brazil) Pectin (Pec) was isolated from orange peels and fully desesterified as reported by Lessa et al (2017) Chitosan (Cs, Mv of 87,000 g/mol and 85

% deacetylated) was purchased from Golden-Shell Biochemical (Yuhuan, China) Magnetite nanopowder (iron (II,III) oxide, 97 % of purity, 50− 100 nm particle size, and magnetization saturation of

91 emu g− 1) was purchased from Sigma-Aldrich (St Louis, MO, USA) Metamizole sodium salt (Mtz, 351.36 g mol− 1) was purchased from Sanofi Aventis Pharma (Bombain, India) Calcium chloride (CaCl2) was purchased from Synth (Diadema, SP, Brazil) All other chemicals were of analytical grade and were utilized without further purification

2.2 Preparation of the magnetic microspheres

Magnetic Pec@Cs microspheres were prepared using a two-step process adapting a methodology described by Rashidzadeh et al (2020) Scheme 1 outlines the microspheres preparation processes Firstly, Pec was completely solubilized in distilled water at a concen-tration of 3 wt-% and magnetite nanoparticles (1 wt-% related to the Pec dry weight) were added The system was homogenized using an ultra-sonic bath (42 kHz for 15 min at 30 ◦C) and transferred to a syringe equipped with a needle (inner diameter of 1 mm) Next, the Pec/mag-netite solution was dropped (speed 1 ml min− 1) into CaCl2 solution (10 wt-%, 20 mL), which was kept under mild orbital stirring (~100 rpm) at room temperature The as-formed microspheres were left to maturate in CaCl2 solution for 15 min After that, the microspheres were recovered

by filtration and thoroughly washed with distilled water to remove the excess of Ca2+ions No release of magnetite was observed during this step

In the sequence, the Pec/magnetite microspheres were put in contact with a Cs solution (1 wt-%, acetic acid solution 1.5 v/v-%, pH 3) under low stirring (~100 rpm) for 2 h at room temperature Lastly, the mi-crospheres coated by Cs were recovered and washed with distilled water and oven-dried (35 ◦C, 24 h) The prepared microspheres were denoted

as mag-Pec@Cs, respectively For comparative and characterization

Scheme 1 The experimental approach used to prepare magnetic-pectin microspheres coated by chitosan

purposes, microspheres without magnetite (denoted as Pec@Cs) and

without the Cs coating (denoted as mag-Pec) were also prepared using

similar procedures

2.3 Drug encapsulation

The preparation of Mtz loaded-microspheres was made using the

same process described in the previous section with minor

modifica-tions Herein, Mtz (1 mg) was added to the Pec or Pec/magnetite

solu-tions before their dripping in the CaCl2 solution It is important to

mention that the amount of Mtz (1 mg) was selected from previous

ex-periments Two sets of Mtz loaded-microspheres were prepared;

Pec@Cs/Mtz and mag-Pec@Cs/Mtz, respectively The Mtz content

encapsulated within the microspheres was determined using a UV–vis

spectrometer (Perkin-Elmer, model Lambda 24, USA) For this, the Mtz-

loaded microspheres (1 g) were completely crushed and soaked in PBS

(0.01 mol L− 1, pH 7.4) for 24 h under stirring The obtained solutions

were centrifuged (5000 rpm for 15 min) and the supernatants were

analyzed by UV–vis spectrometry at λ =271 nm The Mtz content was

estimated using a previously built calibration curve (R2 >0.999) From

these data, the encapsulation efficiency (EE%) and drug loading (DL%)

were calculated per Eq (1) and (2) All samples were analyzed in

triplicate

2.4 Characterization

Photographs of the as-prepared Pec@Cs and mag-Pec@Cs micro-spheres (wet state) were taken with a digital camera (Fig 1a and b) Furthermore, photographs of mag-Pec@Cs microspheres immersed in the aqueous medium were taken in the absence and presence of an external magnet (neodymium permanent magnets, NdFeB, 20 × 10 mm, grade N52) (Fig 1c and d) The average size of the prepared micro-spheres (wet state) was measured using a calibrated digital Vernier caliper micrometer (resolution 0.01 mm) For each microsphere type, the average size was calculated from the data measured from 50 samples chosen randomly Data are expressed as mean ± standard error of the mean

The prepared microspheres were characterized by Fourier Trans-formed Infra-Red (FTIR) spectroscopy, X-ray Diffraction (XRD), Ther-mogravimetric Analysis (TGA), and Scanning Electron Microscopy (SEM) Before the FTIR, XRD, and TG analyses the as-prepared micro-spheres (wet state) were crushed using a mortar and then oven-dried (50 ◦C for 48 h) The powdered samples were sieved before use FTIR spectra were recorded in a Shimadzu (model Affinity) spectrometer (Japan) operating in the region from 4000–400 cm− 1 with a resolution

of 4 cm− 1 and 64 scan acquisitions The samples were blended with KBr and pressed into discs before FTIR analysis XRD diffraction patterns were obtained on a Siemens (model D500) diffractometer (Germany) using Cu-Kα radiation (λ ≈ 1.54 Å), at a tube voltage of 40 kV, and tube current of 30 mA TGA analysis was performed with a Shimadzu (model DTG60) analyzer (Japan) under an N2(g) atmosphere SEM images were recorded using a JEOL (model JSM-6610LV) microscope (USA) Before SEM visualization, the samples were swelled in distilled water, frozen in

N2(l), freeze-dried (-55 ◦C for 48 h) and sputter-coated with gold The liquid uptake capacity was evaluated by swelling experiments

Fig 1 Digital photographs of the as-prepared (a) Pec@Cs and (b) mag-Pec@Cs microspheres The mag-Pec@Cs microspheres immersed in aqueous medium (c) in

the absence and (d) presence of an external magnet

performed in simulated gastric fluid (SGF, pH 1.2) and simulated

in-testinal fluid (SIF, pH 6.8) (Pereira et al., 2013) For this, dry

micro-spheres (50 mg) were put into vials filled with 50 ml of the swelling

medium at room temperature and slow stirring At predetermined

in-tervals, the microspheres were collected, the excess of liquid on their

surfaces was carefully removed, and then, they were weighed again The

swelling ratio at each time interval was calculated per Eq (3):

Swelling(%) =[w s− w d]

w d

where w s is the weight of samples after swelling at a predetermined

interval and w d is the weight at dry state The swelling experiments were

performed in triplicate

The point of zero charge (PZC), a parameter that describes the

con-dition when the electrical charge density on the bead surface is zero, was

estimated from the difference between the initial and final pHs of the

immersion solution (Kosmulski, 2020) Briefly, 200 mg of microspheres

were placed into vials containing NaCl solution (50 mL, 0.1 mol L− 1)

with different pHs (from 2 to 12) The pH was adjusted with HCl or

NaOH solution (0.1 mol L− 1) using a Hannah (model HI2211) pH Meter

(Brazil) The vials were kept under low orbital stirring for 24 h to reach

equilibrium Thus, the microspheres were withdrawn from each vial and

the final pH (pHf) of the solutions was measured immediately The

dif-ference between the initial (pH0) and final pHs (ΔpH = pH0 – pHf) was

plotted against pH0 The pH where the ΔpH is equal to zero was ascribed

as pHPZC

2.5 In vitro release experiments

The Mtz release behavior from the prepared microspheres was

assessed through in vitro experiments using two different media; SGF

(pH 1.2) and SIF (pH 6.8) both without the presence of enzymes (Pereira

et al., 2013) A certain amount of the Mtz-loaded microspheres (200 mg)

were placed into vials filled with 50 ml of the release medium (SGF or

SIF), which were kept at 37 ± 1 ◦C with mild orbital stirring (50 rpm)

over the whole experiment (12 h duration) At predetermined time

in-tervals, stirring was stopped and aliquots (3 mL) were withdrawn,

centrifuged (5000 rpm for 5 min), and spectrophotometrically analyzed

at λ =271 nm An equivalent volume of fresh release medium was

refilled in the system immediately to keep the total volume constant

The cumulative release percentages after each time interval were

calculated per Eq (4) Again, all procedures were done in triplicate

(4)

To verify the effect of an external magnetic field (EMF) on the Mtz

release behavior similar in vitro experiments were carried However, a

permanent cylindrical neodymium permanent magnet (NdFeB,

20 × 10 mm, grade N52) was positioned on the top of the vial containing

the microspheres and the release medium (externally), while another

identical magnet was placed at the bottom Again, SGF and SIF were

used as releasing media At predetermined time intervals, aliquots were

withdrawn from each vial and the amount of Mtz released was estimated

by UV–vis measurements (at λ =271 nm) The cumulative release was

calculated per Eq (4)

3 Results and discussion

3.1 Characterization of the prepared magnetic microspheres

The dripping approach used to prepare the Pec@Cs microspheres

(with and without magnetite) resulted in spherical-like materials as

demonstrated in Fig 1 Microspheres were instantaneously formed after

the dripping of pectin solution into CaCl2 solution due to the ionotropic

gelation between carboxylate groups of pectin and Ca2+ions (Kim et al.,

2017) Next, the pectin-based microspheres were allowed to interact with chitosan, a polycationic polysaccharide, resulting in the coat of the surface of the microspheres Herein, the residual carboxylate groups of pectin interact electrostatically with the amino protonated groups of chitosan The Pec@Cs microspheres exhibited a colorless nature and spherical geometry (Fig 1a) Although the introduction of magnetite did not affect the geometry of the microspheres, the mag-Pec@Cs showed a dark color characteristic of the magnetic nanoparticles embedded into the polymer matrix (Fig 1b) Photographs taken from the prepared mag-Pec@Cs microspheres in aqueous media (Fig 1c) show that they moved toward an external magnetic field (Fig 1d) indicating a suc-cessful magnetization behavior

Table 1 compares the average size and pHPZC data estimated for different prepared microspheres samples As observed, the presence of magnetite in the microsphere formulation decreased their average size

as compared to the bare sample (Pec@Cs) Probably, magnetite nano-particles interact with functional groups distributed along the pectin chains (hydroxyl and carboxyl groups) increasing the crosslinking den-sity within the magnetic microspheres, and thus average size decreases (Kondaveeti et al., 2016) Also, the microspheres coated by the chitosan layer (mag-Pec@Cs and Pec@Cs) exhibited a higher average size sug-gesting the successful deposition of this polysaccharide on the surface of the pectin-based microspheres This is a typical result reported by other studies that use chitosan as a coating agent for different particulate systems (Frank et al., 2020) Overall, the experimental approach used here to prepare microspheres (coated or not) seems to be efficient to obtain microspheres with certain regularity of size and shape It is important to mention that despite the above-discussed features, the average size calculated for these different microspheres systems are statistically similar

The point of zero charge (PZC) is the pH of the suspension at which the net charge on the surface of the microspheres is zero (i.e., [H+] ≈ [OH−]) Generally, the pHPZC value is of great importance since it gives information on pH ranges where the surface of the microsphere is positively or negatively charged (Allouss et al., 2019) Also, this parameter can be useful to investigate the surface charge density of the prepared microspheres According to the data presented in Table 1, mag-Pec exhibits a negatively charged surface at pH conditions higher than 2.83, owing to the carboxylate groups of pectin Thus, at pH 3 (experimental condition) the surface of these microspheres is ready to interact electrostatically with the cationic chains of chitosan Indeed, the chitosan-coated microspheres (mag-Pec@Cs and Pec@Cs) exhibited higher pHPZC values, confirming the coating process Due to the chitosan layer, the pH range where the surface of the microspheres is negatively charged is shortened Additionally, the pHPZC estimation suggests that magnetite does not affect the surface charge of the prepared micro-spheres, probably because it remains embedded within the pectin core SEM images recorded from the mag-Pec, mag-Pec@Cs, and Pec@Cs microspheres were used to investigate their morphology and micro-structure As shown in Fig 2, all microsphere samples exhibited a spherical-like shape with different levels of roughness and cracks Ac-cording to Jeddi & Mahkam (2019), the cracks appear due to the drying process and can be ascribed to the high volume of water inside the polymer matrices The SEM images of mag-Pec (Fig 2a and b) show that this sample has a more uniform and compact surface, which strengthens the suggestion that magnetite increased the crosslinking density of the

Table 1

Average size and pHPZC values estimated for different microspheres samples

a The average size was calculated from wet microspheres

pectin matrix Besides, a denser polymer matrix retains a smaller volume

of water, which may explain the lower cracking on its surface At higher

magnification (Fig 2b) it can be observed that the mag-Pec microsphere

has a highly rough and irregular surface, with polyhedric particles of

variable sizes In contrast, SEM images of the mag-Pec@Cs and Pec@Cs

(Fig 2c–f) revealed that the chitosan coating increased the cracks on the

surface of the microspheres, while it reduced the surface roughness

Similar reports are done by other authors that have utilized chitosan as a

coating agent for microspheres (Finotelli et al., 2010; Rashidzadeh et al.,

2020) Comparing mag-Pec@Cs and Pec@Cs, their morphologies are

quite similar indicating that magnetite nanoparticles embedded on the

pectin core exert a negligible effect on microspheres surfaces

FTIR spectroscopy was used to evaluate the microsphere formation

and chitosan-coating process All obtained spectra are shown in Fig 3a

The spectrum of raw pectin exhibited a broad band centered at

3418 cm− 1 due to O–H stretching (hydroxyl groups) and other

char-acteristic bands at 2930 cm− 1, 1642 cm− 1, and 1421 cm− 1 ascribed to

C–H stretching (CHx groups) and asymmetric and symmetric C––O

stretching (carboxyl groups) (Lessa et al., 2017) The bands at

1157 cm− 1, 1100 cm− 1, and 1035 cm− 1 are due to C–O–C stretching

(glycosidic bond, ring) and C–C/C–O stretching (Demir et al., 2020)

After the mag-Pec formation, the bands associated with the hydroxyl

and carboxyl groups of pectin were shifted to different wavenumber due

to the bind of such groups to Ca2+ions (Lessa et al., 2017) Moreover,

the Ca2+affects the electrostatic environment around the functional groups of pectin causing changes in the intensity of multiple bands compared to the spectrum of raw pectin For example, the band ascribed

to O–H stretching is sharpened and its center is moved to 3422 cm− 1, while the band ascribed to asymmetric C––O stretching is shifted to 1630

cm− 1 Similar results concerning this kind of microspheres were re-ported in the literature (Assifaoui et al., 2010; Lessa et al., 2017) Also, the appearance of a new band at 554 cm− 1 can be associated with the Fe–O bond, indicating the successful entrapment of magnetite nano-particles on the pectin-based microspheres (Marin et al., 2018) FTIR spectrum of raw chitosan exhibited a broad band centered at 3402 cm− 1

due to O–H and N–H stretching (hydroxyl and amine groups) and bands at 2901 cm− 1, 1638 cm− 1, 1570 cm− 1, and 1235 cm− 1 corre-sponding to C–H stretching (CH3 groups), C––O stretching (amide I), N–H bending (amide II), and C–N stretching (amide III) (Brugnerotto

et al., 2001) Bands at 1163 cm− 1 and 1082 cm− 1 are due to C–C and C–O stretching related to the saccharide structure of chitosan ( Gonza-lez-Pabon et al., 2019) After the coating of the mag-Pec microspheres with chitosan, some discrepancies were noticed in the spectrum ob-tained for mag-Pec@Cs The bands associated with the carboxyl groups

of pectin were shifted to 1628 cm− 1, while the bands corresponding to amino groups of chitosan were reduced in intensity and shifted to

1552 cm− 1, respectively The shifting of these bands to lower wave-number regions is caused by the electrostatic interaction among the

Fig 2 Images obtained by SEM from dried (a,b) mag-Pec, (c,d) mag-Pec@Cs and (e,f) Pec@Cs microspheres

–COO− groups of pectin and –NH3+groups of chitosan The absence of

new bands strengthens the suggestion that only electrostatic interactions

occur between the polysaccharides Similar results were reported to

authors that used chitosan to coat microspheres based on alginate, a

carboxyl-rich polysaccharide (Jeddi & Mahkam, 2019; Rashidzadeh

et al., 2020) It is important to note that the band associated with the

magnetite is still observed in the mag-Pec@Cs spectrum Finally, as

shown in Fig 3a, the spectrum of the Pec@Cs microspheres showed to

be similar to mag-Pec@Cs indicating that the presence of magnetite does

not affect the electrostatic interaction between pectin and chitosan

Fig 3b shows the XRD patterns obtained for raw pectin and chitosan

and Pec@Cs and mag-Pec@Cs microspheres As observed, the XRD

pattern of pectin exhibited some diffraction peaks at 2θ ≈ 12.7◦, 20.5◦,

26.2◦, and 30.1◦ indicating that this polysaccharide has some

crystal-linity (Kumar & Chauhan, 2010) Probably, crystalline regions are

formed as a result of intra and intermolecular hydrogen bonds among

the pectin chains For chitosan, it was observed a typical broad

diffraction peak at 2θ ≈ 20.3◦ indicating its semi-crystalline nature

(Lessa et al., 2018) The XRD pattern obtained for the Pec@Cs

micro-spheres did not exhibit any diffraction peak indicating the prevalence of

amorphous structure Indeed, the electrostatic interaction between the

pectin-Ca2+ions and pectin-chitosan disrupts the crystalline regions in

the raw polysaccharides, explaining the amorphous nature of Pec@Cs

microspheres In contrast, the XRD pattern of mag-Pec@Cs microspheres

exhibited diffraction peaks at 2θ ≈ 30.2◦, 35.7◦, 43.3◦, 57.2◦, and 62.7◦,

which correspond to the typical reflection planes of cubic Fe3O4

nanoparticles, with following corresponding indices (220), (311), (400), (511), and (440) (JCPDS number #19-0629) (Dar & Shivashankar,

2014) The presence of these diffraction peaks confirms the entrapment

of magnetite into the microspheres without changing its structure (Xiao

et al., 2011) Besides, the absence of new diffraction peaks compared to the bare microspheres (Pec@Cs) suggests the magnetite nanoparticles did not affect the polymer matrix ordering

TGA/DTG analysis was performed to evaluate the thermal behavior

of the prepared microspheres and results are shown in Fig 4a and b TGA curve of raw pectin exhibited two weight loss stages, where the first (between 30 and 125 ◦C) caused a weight loss of 15 % due to the evaporation of water The second stage (between 195 and 290 ◦C, with a maximum at 241 ◦C) is due to the thermal depolymerization of the pectin backbone resulted in a weight loss of 43 % (Lessa et al., 2017) At

500 ◦C, the residual weight of pectin was around 42 % Similarly, raw chitosan exhibited two main weight loss stages The first weight loss around of 10 % (between 30 and 130 ◦C) was due to the evaporation of adsorbed water, while the second weight loss stage (between 230 and

400 ◦C, with a maximum at 303 ◦C) was attributed to the thermal decomposition and deacetylation of chitosan backbone (Nam et al.,

2010) For chitosan, the residual weight at 500 ◦C was around 43 % For the microspheres (Pec@Cs and mag-Pec@Cs), TGA curves were quite similar; however, some discrepancies can be noticed In summary, both curves exhibited three main weight loss stages In the first stages (be-tween 30 and 120 ◦C), Pec@Cs lost around 17 % of weight, while mag-Pec@Cs around 21 % due to the water evaporation This data re-veals that the entrapment of magnetite into the pectin matrix increased the water content into the magnetic microsphere compared to the bare sample It is worthy to point out that both microspheres samples were thoroughly dried under identical conditions (up to constant weight) before TGA analysis Moreover, comparable finds were also reported by

Jeddi & Mahkam (2019) The second and third stages were observed between 210 and 350 ◦C and are attributed to the thermal decomposi-tion of each polysaccharide For Pec@Cs, the maximum temperatures for pectin and chitosan decomposition were found to be at 257 ◦C and

303 ◦C and the total weight loss was around 25 % For mag-Pec@Cs, the maximum temperatures were found to be 251 ◦C and 302 ◦C, while the weight loss was around 29 % This result suggests the presence of magnetite has a slightly negative effect on the thermal stability of the mag-Pec@Cs microspheres Additionally, at 500 ◦C it was found that the residual weight of Pec@Cs was higher than that observed for mag-Pec@Cs Probably, the magnetite nanoparticles catalyzed the thermal decomposition of pectin/chitosan chains explaining the ob-tained results Indeed, some papers have described the ability of metal oxides like to Fe3O4 to accelerate the thermal decomposition of poly-saccharides (Jurikova et al., 2012; Ziegler-Borowska et al., 2016) The liquid uptake is an essential property of hydrophilic materials and paramount for functional DDS Herein, the liquid uptake capacity of the prepared microspheres was evaluated by swelling experiments per-formed in SGF (pH 1.2) and SIF (pH 6.8) The swelling curves built for Pec@Cs and mag-Pec@Cs are shown in Fig 5a and b Both microspheres swelled quickly in SGF achieving high swelling rates before 30 min For Pec@Cs, the swelling rate seems to slow down after 20− 25 min and, then, the equilibrium is achieved close to 60 min Next, the swelling tends to level off until the end of the experiment The maximum swelling rate calculated for this sample in SGF was around 233 % Conversely, mag-Pec@Cs exhibited a slightly faster initial swelling achieving the equilibrium sooner than the bare microspheres (ca 30 min) For these microspheres, the maximum swelling rate was around 275 % In general lines, Pec@Cs and mag-Pec@Cs showed a high swelling performance, which can be explained by the acidic condition of SGF that affects the charge of the different functional groups Under this pH condition, the amino groups in chitosan and carboxyl groups in pectin are both pro-tonated As a result, the electrostatic interaction between pectin and chitosan decreases, as well as the pectin-Ca2+ interactions (Lofgren

et al., 2002) Simultaneously, the repulsive forces among the protonated

Fig 3 (a) FTIR spectra recorded from raw pectin and chitosan and prepared

microspheres (mag-Pec, mag-Pec@Cs, and Pec@Cs) (b) XRD patterns of raw

pectin and chitosan and prepared microspheres (Pec@Cs and mag-Pec@Cs)

amino groups in chitosan increase Thus, the polymer matrix expands

allowing that a high amount of liquid moves inward the microspheres It

is important to inform that the hydrophilic nature of both

poly-saccharides enhances the liquid uptake capacity of the prepared

mi-crospheres The data depicted in Fig 5a also reveals that mag-Pec@Cs

microspheres have a higher liquid uptake capacity than Pec@Cs In

practical terms, the addition of 1 wt-% of magnetite allowed to increase

the maximum swelling by 42 % Probably, the presence of magnetite

nanoparticles impaired the ionotropic crosslinking of pectin chains by

Ca2+ions reducing the crosslinking density within the microspheres

Overall, a lower crosslinked density favors the water uptake process (Bueno et al., 2013) Moreover, such impairment caused by magnetite in the ionotropic crosslinking can also explain the lower thermal stability

of mag-Pec@Cs, as observed from TGA/DTG analysis

In SIF (pH 6.8), the liquid uptake capacity of both microspheres was noticeably lower than in SGF, as shown in Fig 5b This trend highlights that the prepared microspheres are exceedingly sensitive to pH varia-tions Under neutral pH, the mag-Pec@Cs microspheres showed again a faster swelling profile compared to the Pec@Cs However, at this pH condition, the swelling equilibrium was achieved faster than in acidic conditions (before 10 min) The maximum swelling rate was calculated

to be 68 % and 180 % for Pec@Cs and mag-Pec@Cs, respectively At pH 6.8, the carboxyl groups in pectin and amino groups in chitosan are deprotonated, which increases the interaction between pectin chains and Ca2+ions At the same time, the electrostatic interactions between pectin and chitosan decrease However, the chitosan coat probably re-mains on the surface of microspheres since hydrogen bonds can be formed between the polysaccharides Furthermore, this suggestion is strengthened by the low solubility of chitosan in neutral and alkaline pH conditions (Nie et al., 2016) As demonstrated by these swelling ex-periments, the pH-sensitive properties of Pec@Cs and mag-Pec@Cs can

be attractive to trigger and control the release of encapsulated bioactive compounds like drugs, for example

3.2 Release experiments

In vitro experiments were conducted to investigate the release ability

of the prepared microspheres using Mtz a model drug Earlier to the release experiments, the encapsulation efficiency (EE%) and drug loading (DL%) were estimated For Pec@Cs/Mtz, EE% and DL% were calculated to be 85 ± 1 % and 0.14 ± 0.02 %, while mag-Pec@Cs/Mtz showed EE% and DL% equal to 88 ± 2 % and 0.15 ± 0.04 %, respec-tively From a statistical viewpoint, the results concerning both micro-sphere samples are similar However, it can be mentioned that both microspheres showed EE% values higher than 85 %, indicating a mini-mal loss of Mtz during the encapsulation process

Fig 6a and b show the release profile of Mtz from Pec@Cs/Mtz mag- Pec@Cs/Mtz in SGF (pH 1.2) and SIF (pH 7.4) at 37 ◦C Moreover, additional release experiments were performed with the loaded mag-netic microspheres using an external magmag-netic field (EMF) to evaluate its effect on the Mtz release In SGF, the drug release occurred quickly during the first hour of the experiment for all tested samples, mainly for the microspheres exposed to EMF Next, the release process slows down and remained constant until the end of the experiment After 12 h, the percentages of Mtz released from Pec@Cs/Mtz, mag-Pec@Cs/Mtz, and mag-Pec@Cs/Mtz (with EMF) in SGF were calculated to be around 18 %,

21 %, and 26 %, respectively These results seem to be inconsistent with

Fig 4 (a) TGA and (b) DTG curves obtained for raw pectin and chitosan and prepared microspheres (mag-Pec, mag-Pec@Cs, and Pec@Cs)

Fig 5 Swelling profile of Pec@Cs and mag-Pec@Cs microspheres in (a) SGF

(pH 1.2) and (b) SIF (pH 6.8) at 37 ◦C

the swelling data that demonstrated that under acidic conditions both

Pec@Cs/Mtz and mag-Pec@Cs/Mtz microspheres exhibit high liquid

uptake capacities To explain these results, it should be noticed that the

Mtz molecule contains negatively charged groups that can interact with

the chitosan coat that under acidic conditions is positively charged (due

to its protonated amino groups) Similar results were reported by Bhise

et al (2008) and Sun et al (2010) that designed DDS based on chitosan

for sustained release of anionic drugs such as naproxen and enoxaparin

According to the authors, the interactions between cationic chitosan and

the anionic drugs form stable systems from which the drugs are released

over a more prolonged time interval These finds corroborate the high

values of log P calculated for Mtz under these release conditions (log

P ≥ 3.05) It is important to mention that the cationic nature of the

chitosan coat under acidic conditions also can be ranked as an additional

advantage since it is responsible for mucoadhesion via ionic interaction

with the mucus of the gastric system (Shafabakhsh et al., 2020)

Results depicted in Fig 6a also reveal that the presence of an EMF

increases the Mtz release rate from mag-Pec@Cs and promotes a gain of

5 % in the cumulative amount released after 12 h compared to the

conventional release (i.e., without EMF) This find confirms that mag-

Pec@Cs show magnetic-responsible behavior As explained by

Rashid-zadeh et al (2020), the magnetic nanoparticles embedded into the

mi-crosphere’s matrix are agitated and moved under the influence of EMF,

which leads to the relaxing of polymer chains Thus, this relaxation

phenomenon may have led to mechanical deformation and subsequent

tensile stresses, resulting in an enhancement in the amount of drug

released (Paulino et al., 2012; Rashidzadeh et al., 2020) Additionally,

under EMF the magnetic nanoparticles are aligned within the

micro-spheres decreasing the barrier effect against the drug release process

(Marin et al., 2018)

The Mtz release from the Pec@Cs/Mtz and mag-Pec@Cs/Mtz in SIF

showed a similar profile compared to SGF media (Fig 6b) Overall, the

drug was released quickly at the beginning of the experiment, and, then,

the release process slows down as time goes on However, after 12 h the

amount of Mtz released from the microspheres is markedly higher than

that estimated in SGF Herein, the percentages of Mtz released from

Pec@Cs/Mtz, mag-Pec@Cs/Mtz, and mag-Pec@Cs/Mtz (with EMF)

after 12 h were calculated to be around 71 %, 75 %, and 91 %,

respectively These results can be explained by the absence of charges in

the chitosan layer under neutral conditions (i.e., absence of interaction

with Mtz molecules) Besides, at pH 6.8 the carboxylic groups in pectin

are deprotonated increasing the negatively repulsive forces with the

anionic Mtz, thus, favoring the release Hence, the calculated values of

log P (≤ 1.04) were noticeably lower than those calculated for Mtz in

SGF Furthermore, under EMF the drug release process was enhanced

again The Mtz release increased by 16 % after 12 h compared to the

experiment without EMF In summary, the release experiments indicate that both microspheres (Pec@Cs/Mtz, mag-Pec@Cs/Mtz) are sensitive

to pH, while mag-Pec@Cs/Mtz is simultaneously sensitive to EMF

To gain insights about the release process and mechanism, all results shown in Fig 6 were fitted by different mathematical models of drug release Herein, Higuchi, Korsmeyer-Peppas, and Weibull models were utilized The Higuchi model (Eq (5)) is often used for the assessment of drug release from polymeric matrices via diffusion-controlled processes (Mircioiu et al., 2019) Korsmeyer-Peppas is a semi-empirical model (Eq

(6)) generally used to analyze drug release when the mechanism is not well known or multiple mechanisms are involved (Korsmeyer et al.,

1983) Moreover, this model enables only fitting the data related to the first 60 % of drug release Finally, Weibull is an empirical model (Eq

(7)) frequently used to analyze the drug release from micro and nano-particles in different experimental conditions (Ignacio et al., 2017)

Herein, M t refers to the amount of cumulative drug released at each time

(t), M∞ is the amount of cumulative drug release at infinite time, k H and

k KP are the Higuchi and Korsmeyer-Peppas constants, and n is the release

exponent associated with the drug release mechanism Furthermore, in

Eq (7), the parameters a and b are the "scale" and "shape" factors in the

Weibull distribution (Ignacio et al., 2017) The fitting parameters ob-tained from the mathematical models are summarized in Table 2 Analyzing the coefficients of determination (R2) given in Table 2, it is observed that the highest R2 values were obtained for the Weibull model, indicating that this model adjusts well to the experimental data Indeed, the Weibull model had the best fit for all tested samples and

conditions In this context, the parameter b ("shape" factor) can be used

as an indicator of the mechanism of transport for the drug through the

polymeric matrix Generally, a value of b < 0.75 denotes Fickian diffu-sion, while a value in the range 0.75 < b < 1.0 denotes a combined

mechanism (Fickian diffusion and swelling-controlled transport) Values

of b > 1 are associated with a complex transport/release mechanism (i

e., a combination of different mechanisms such as erosion, diffusion, and swelling) (Mircioiu et al., 2019) From Table 2, it is noticed that Mtz release from Pec@Cs and mag-Pec@Cs microspheres change according

to the release media In SGF, the release mechanism is guided by Fickian diffusion, while in SIF it changes to a combined mechanism (Fickian diffusion and swelling-controlled transport) Curiously, the presence of

an EMF does not affect the release mechanism of mag-Pec@Cs It means

Fig 6 In vitro Mtz release profile from Pec@Cs/Mtz and mag-Pec@Cs/Mtz microspheres in (a) SGF (pH 1.2) and (b) SIF (pH 6.8) at 37 ◦C For mag-Pec@Cs/Mtz the release experiments were performed in the absence and presence of an external magnetic field (EMF)

that the electrostatic interactions between the polysaccharides exert a

higher effect on the drug release process than the presence of an EMF

This find corroborates other similar studies focused on the use of

mag-netic polymeric systems as DDS (Demarchi et al., 2014; Uva et al., 2015)

From the Weibull model, the values of a ("scale" factor) can be used to

calculate the T d parameter [a = (T d ) b], which corresponding to the time

required to release 63.2 % of the encapsulated drug (Barboza et al.,

2014) The values of T d obtained reveal that the presence of EMF

allowed an accelerated Mtz release from mag-Pec@Cs in both tested

media

4 Conclusions

Here, we succeed in preparing magnetic microspheres based on

pectin/magnetite coated by chitosan The microspheres were obtained

by ionotropic gelation of pectin with Ca2+ ions followed by

poly-electrolyte complexation of a chitosan coating According to FTIR and

XRD, the magnetite nanoparticles were entrapped into the

micro-spheres SEM and TGA/DTG analyses showed that magnetite caused

changes in the morphology and thermal stability of the microspheres

Moreover, these characterization analyses confirmed the coating of the

pectin/magnetite microspheres by chitosan Swelling experiments

per-formed in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal

fluid (SIF, pH 6.8) revealed that the presence of magnetite enhances the

liquid uptake capacity of the microspheres in both media Additionally,

metamizole (Mtz) was efficiently encapsulated into the magnetic

mi-crospheres, and in vitro release experiments were performed in SGF and

SIF The results showed that the release process can be adjusted by

varying the pH of the medium and it is favored in SIF Weibull model

better fitted the release data, indicating that the release mechanism is

guided by Fickian diffusion in SGF, while in the SIF medium it changes

to a combined mechanism (Fickian diffusion and swelling-controlled

transport) Release experiments in the presence of an external

mag-netic field (EMF) showed that the drug release is boosted in both release

media suggesting that the microspheres prepared in this study also exhibit a magnetic-sensitivity property Based on our finds, the magnetic microspheres can be considered potential candidates for drug delivery applications, particularly in colon-localized delivery or in cancer ther-apy (tumor inhibition)

CRediT authorship contribution statement Thalia S.A Lemos: Methodology, Formal analysis, Investigation, Writing - original draft Jaqueline F de Souza: Methodology, Formal analysis, Investigation Andr´e R Fajardo: Supervision, Project

admin-istration, Writing - review & editing

Declaration of Competing Interest

The authors report no declarations of interest

Acknowledgments

The authors are thankful to CNPq (Process 404744/2018-4) for financial support A.R.F also thanks CNPq for his PQ fellowship (Process 303872/2019-5) This study was financed in part by the Coordenaç˜ao de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES/Proap), Finance Code 001

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