Materials Science & Engineering C 118 (2021) 111412

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Materials Science & Engineering C

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Development and evaluation of naproxen-loaded sericin/alginate beads for T

delayed and extended drug release using different covalent crosslinking
agents
Emanuelle D. Freitasa, Vitória M.S. Freitasa, Paulo C.P. Rosab, Meuris G.C. da Silvaa,
Melissa G.A. Vieiraa,
⁎

a
School of Chemical Engineering, University of Campinas – UNICAMP, Albert Einstein Avenue, 500, 13083-852 Campinas, SP, Brazil
b
Faculty of Pharmaceutical Sciences, University of Campinas – UNICAMP, Cândido Portinari Street, 13083-871 Campinas, SP, Brazil

ARTICLE INFO ABSTRACT

Keywords: Different polymer matrix compositions based on sericin and alginate blend (using or not the covalent cross­
Sericin/alginate blend linking agents dibasic sodium phosphate, polyvinyl alcohol and polyethylene glycol) were evaluated to entrap
Naproxen naproxen. Sericin has been shown to be essential for improving incorporation efficiency. Comparing the for­
Extended release formulation mulations with and without crosslinking agent, the best results were obtained for that composed only of sericin
and alginate, with satisfactory values of entrapment efficiency (> 80%) and drug loading capacity (> 20%). In
this case, delayed release (< 10% in acid medium) and prolonged release (~360 min) were achieved, with a
complex release mechanism involving swelling and polymer chain relaxation. The incorporation of the drug
could be confirmed by the techniques of characterization of X-ray diffraction (XRD), scanning electron micro­
scopy (SEM) and Fourier transform infrared spectroscopy (FTIR), as well as drug compatibility with the polymer
matrix. In addition, particles of suitable size for multiparticulate systems were obtained and with higher thermal
stability when compared to the pure drug.

1. Introduction process of degumming the silk fiber [4]. However, sericin's bio­
compatibility and biodegradability have led to increased interest in this
Conventional release pharmaceutical forms have contributed sig­ protein for application in the fields of biomedicine, cosmetics and
nificantly to the treatment of diseases over the past decades. However, pharmaceutical [5], being reported studies on the incorporation of re­
in recent years it has been developed new pharmaceutical forms tai­ sveratrol [6] and atorvastatin [7] into sericin nanoparticles. One of the
lored for specific cargo and release at specific sites [1]. By modifying great advantages of sericin is its chemical structure, which enables
the release of a drug, it is possible to improve drug therapy, by reducing crosslinking, polymerization or blending with other polymers, gen­
side effects and increasing its desired effects, besides being more con­ erating compounds with improved properties [8]. Polyvinyl alcohol
venient than the costly and long-term process of developing new drugs (PVA) [9], collagen [10] and alginate [11] can be mentioned among the
[2]. In this context, the use of polymers to obtain new drug delivery various polymers used in composition with sericin.
systems is very common. Polymers of natural or synthetic origin have Alginates are natural polysaccharides obtained from brown algae in
good flexibility and can be modified to obtain versatile properties and, the form of alginic acid, which is converted to a salt, usually sodium
consequently, the desired controlled release profile. Among the most alginate. Alginates are formed by alternating or random subunits of L-
common natural polymers for application in this field, cellulose and its guluronic and D-mannuronic acids, G and M, that directly affect their
derivatives stand out, in addition to proteins and other polysaccharides properties, such as their strength [12]. This polymer has been ex­
[3]. tensively used in pharmacological applications especially for its low
Sericin, a globular protein present in cocoons of the Bombyx mori toxicity and biocompatibility. In addition, in the presence of divalent
silkworm, acts as an adhesive binder, maintaining the structure of the ions, as Ca2+, it undergoes ionic crosslinking, forming an ionotropic
fiber and cocoon. For many years, these cocoons have been used only to hydrogel due to intermolecular bonds [13]. Several articles report al­
provide silk for the textile industry, with sericin being discarded in the ginate as matrix for drug incorporation, e.g. in the case of roflumilast

⁎
Corresponding author.
E-mail address: melissagav@[Link] (M.G.A. Vieira).

[Link]
Received 27 August 2019; Received in revised form 10 August 2020; Accepted 11 August 2020
Available online 22 August 2020
0928-4931/ © 2020 Elsevier B.V. All rights reserved.
E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

[14], retinoic acid [15], metformin hydrochloride [16] and pantothenic 2. Experimental
acid [17].
The blend of sericin and alginate has been successfully proposed for 2.1. Material
drugs entrapment as diclofenac sodium [18] and ibuprofen [19] in
order to modify their release. Like the mentioned drugs, naproxen is a Sericin was extracted from Bombyx mori silkworm cocoons, nicely
propionic acid derivative of the non-steroidal anti-inflammatory drugs. supplied by The Bratac Silk Mills Company (Londrina-PR, Brazil).
It has anti-inflammatory and analgesic properties and has been in­ Naproxen (Purifarma, São Paulo-SP, Brazil), sodium alginate (Sigma-
dicated for rheumatoid arthritis treatment because it is as efficient as Aldrich, St Louis-MO, USA), CaCl2, HCl (Anidrol, Diadema - SP, Brazil),
aspirin and has a better tolerability [20]. The problem of naproxen is NaH2PO4 and NaOH (Dinâmica, Diadema - SP, Brazil) were commer­
related to its side effects, which can cause bleeding, ulceration or per­ cially obtained. For the preparation of all solutions, ultrapure water was
foration in the gastric environment when ingested. Thus, it becomes an used (Reverse Osmosis, Gehaka, Brazil).
excellent candidate for obtaining prolonged or controlled drug release,
improving its bioavailability [21]. 2.2. Extraction of sericin and concentration of sericin solution
It is possible that a certain drug release goal will not be met by using
the pure blend of sericin and alginate, as it was observed by Freitas Sericin extraction was carried out following the methodology of
et al. [22]. In that case, an additional process may be necessary, such as Silva et al. [39]. The dirt of the cocoons was removed and they were cut
chemical crosslinking, which seeks to produce more resistant pharma­ into small pieces (~1 cm2) by hand, so they were rinsed with tap water
ceutical particles and improve incorporation and dissolution results, and ultrapure water. The small pieces, oven dried at 50 °C for about
through the addition of a covalent crosslinking agent. This is possible 12 h, were immersed in 4:100 (w/V) ultrapure water for the auto­
because a permanent hydrogel is formed, due to the irreversible che­ claving degumming stage (AV-18, Phoenix, Brazil), at 1 kgf cm−2
mical nature of the covalent bond, improving the mechanical properties (98,066.5 Pa) and 120 °C (393 K), for 40 min. The sericin solution was
of the gel [23,24]. The crosslinking of natural polymers with different isolated by simple filtration and kept in a closed bottle during 24 h at
covalent crosslinking agents has been extensively reported, such as PVA room temperature. It was possible to observe the stabilization of the
as crosslinking agent of alginate [25] and sericin [26], polyethylene protein granules of greater molar mass. To increase the concentration of
glycol (PEG) as alginate [27] and sericin crosslinker [28] and dibasic the sericin solution, the freezing/thawing methodology was used, as
sodium phosphate (DSP) as cellulose [29] and chitosan crosslinker reported by Da Silva et al. [40]. Thus, after stabilization at room tem­
[30]. The addition of crosslinkers has typically been involved in the perature, the solution freezed for at least 24 h and then it was thawed at
construction of a three-dimensional network without changing the room temperature. Sericin of higher molar mass was filtered and au­
functionality of the polymer matrix. However, discoveries have been toclaved to dissolve the protein granules (1 kgf cm−2, 120 °C, 10 min)
made evidencing the variation of properties of materials by the addition and finally diluted with ultrapure water 2.5% (w/V).
of crosslinking agents, confirming the possibility of obtaining better
results in the present work [31]. 2.3. Drug incorporation and particles preparation
PVA is a synthetic hydrophilic polymer that has been used in drug
applications especially for its non-toxicity and degradability [25,32]. The incorporation of the drug and the production of particles fol­
PEG is also a synthetic hydrophilic polymer with biocompatibility lowed the methodology of Freitas et al. [19], modified from Khandai
properties and which is widely used in pharmaceutical applications et al. [41]. The sericin solution (2.5%, w/V) was autoclaved and then
[33]. DSP is a reagent used to obtain a biodegradable and biocompa­ shaken in Ultraturrax™ (T18, IKA, USA) at 4000 rpm until it reached
tible drug release form, in addition to presenting low cost [34]. Thus, in 55 °C (328 K). The next step was the addition of sodium alginate, under
the present work the production of naproxen particles was proposed, stirring of 4000 rpm until homogenization and then naproxen, under
using the sericin and alginate blend as polymer matrix, in order to stirring of 4000 rpm and 8000 rpm subsequently, until homogenization.
obtain modified release forms. It is common studies that evaluate the Then, when appropriate, PVA/PEG/DSP was added, under the same
incorporation of naproxen into polymeric matrices aiming to modify its stirring conditions as used above. Concentrations of sodium alginate,
release, as is the case of the methoxy poly(ethylene glycol)–poly(ε-ca­ 2.8% (w/V), and naproxen, 2.0% (w/V), followed results reported by
prolactone) (mPEG–PCL) copolymer [35] and the locust bean gum Vidart et al. [18] to obtain high incorporation efficiency and prolonged
polymeric matrix [36]. In addition, researches that evaluates the in­ release of drug. The concentration of the crosslinking agent, 2.0% (w/
corporation of naproxen into matrices involving sericin or alginate are V), was based on the incorporation tests reported by Freitas et al. [23]
also reported, e.g. sericin/poly(γ-glutamic acid) hydrogel [37] and al­ for ketoprofen incorporation. In the present paper, formulations were
ginate/oligochitosan/Eudragit® L100-55 microparticles [38]. However, developed using PVA (NPVA), DSP (NDSP) and PEG (NPEG), in addi­
the specific case of the sericin/alginate matrix was not reported, in­ tion to formulations without crosslinking agent (NSER) and without
dicating the novelty of the present study. Furthermore, the present sericin (NALG).
manuscript proposes to evaluate this matrix using different covalent The production of the particles was based on the blend ionic gelling
crosslinking agents, which could improve its incorporation and release property in the presence of divalent ions. Thus, the blend with the drug
properties. embedded was dripped in CaCl2 solution (3%, w/V) under constant
Formulations were developed with alginate only, only with the magnetic stirring with the aid of a peristaltic pump (77201-60,
blend and also with the addition of PVA, PEG and DSP as covalent Masterflex L/S, USA). At the end of the dripping, the gelled particles
crosslinking agents. To verify the best formulations obtained, the en­ were maintained at 100 rpm for 30 min in jar test. The particles were
trapment efficiency, drug loading and dissolution in simulated enteric then rinsed with tap water and ultrapure water and subsequently kept
medium were evaluated. Analytical characterization techniques were to dry at room temperature.
used to evaluate the crystallinity of the particles (XRD), their mor­
phology (SEM), functional groups (FTIR), size and size distribution 2.4. Characterization of drug-loaded beads
(Optical Microscopy - OM), and thermal stability (Thermogravimetry -
TG/DTG and Differential Thermal Analysis - DTA). 2.4.1. Determination of drug loading capacity and entrapment efficiency
Sericin and alginate particles have demonstrated complete dissolu­
tion in phosphate buffer (pH 6.8) after 24 h [42]. Thus, to determine
the entrapment efficiency (EE) and the drug loading capacity (DL), it
was followed the methodology proposed by Sinha et al. [43], who

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

evaluated alginate-okra particles for incorporation of diclofenac so­ where kH is the Higuchi release constant. The assumptions of Eq. (5)
dium. 100 mg of particles were contacted with 500 mL of buffer solu­ involve neglecting matrix swelling or dissolution and edge effects
tion for about 24 h, followed by sonication (LS-9.5DA, LimpSonic, (unidirectional diffusion), the initial drug concentration much greater
Brazil). The suspension was filter through a qualitative filter paper. The than its solubility, and the conditions of the sink being perfect [49,50].
concentration of naproxen was determined by UV–vis spectroscopy Weibull model (Eq. (6))
(UVmini1240, Shimadzu, Japan) at 332 nm. The assays were performed It is an empirical model and, therefore, without kinetic funda­
in triplicate and Eqs. (1) and (2) were used to EE and DL, respectively. mentals, it is not possible to obtain any information about the proper­
ties of dissolution kinetics.
Actual drug content in beads
EE (%) = . 100
Theoretical drug content in beads (1) (t Ti )b
Q = Q0. 1 exp
Weight of drug in beads
a (6)
DL (%) = . 100
Weight of beads (2) Nevertheless, this model allows describing the dissolution curve in
terms of applicable parameters. The Ti (location parameter) refers to lag
where Actual drug content in beads was determined by spectroscopy and
time prior to the initiation of drug release (generally equal to zero). The
Theoretical drug content in beads was measured considering the amount
b (shape parameter) characterizes the type of curve as parabolic, with
of drug initially added. All the obtained results were submitted to the
greater initial slope and then consistent with exponential (b < 1),
Tukey Test, using the software Statistica™, to evaluate if they are sta­
exponential (b = 1), or sigmoid, with upward curvature and followed
tistically equivalent.
by turning point (b > 1). The a (scale parameter) can be better re­
placed by the Td parameter (a = (Td)b), which represents the time re­
2.4.2. In vitro drug release study quired to 63.2% drug release.
To evaluate the naproxen release, in vitro assays were performed Korsmeyer-Peppas model (Eq. (7))
using simulated gastrointestinal tract media. For the study of drug re­ To evaluate the mechanism of drug release, it is possible to employ
lease, U. S. Pharmacopoeia apparatus 1 (basket) was used, at 50 rpm this empirical model, adjusting the data of the first 60% of drug re­
and 37.0 ± 0.5 °C. An amount of particles corresponding to 110 mg of leased.
naproxen was added into the basket, considering the recommended
dosage of 220 mg for prolonged naproxen release. The gastroresistant Q/ Q = kKP . t n (7)
characteristic was evaluated for delayed release dosage forms using, for Q/Q∞ is the drug fraction released at time t, kKP is the release rate
2 h, 1000 mL of 0.1 M HCl solution. Prolonged release of the for­ constant and n is the release exponent. The value of n allows inferring
mulations was assessed by dissolution in pH 7.4 phosphate buffer so­ the mechanism involved in the drug release: for cylindrical shape, for
lution (900 mL) for 8 h, with aliquot removal at predetermined times. n ≤ 0.45, Fickian Diffusion, 0.45 < n < 0.89, Anomalous (non-
The naproxen concentration was determined by UV–vis spectroscopy Fickian) transport, for n = 0.89, Case-II transport, and for n > 0.89,
[44]. Super Case-II transport. For spherical shape, 0.43 instead of 0.45, and
0.85 instead of 0.89 [51].
2.4.3. Mathematical modeling of drug release Hopfenberg model (Eq. (8))
Release of drugs from polymer matrices can occur by three main To evaluate the release mechanism, this model can also be used, as
processes, namely, by diffusion of the drug through the non-degraded it was developed for erodible polymer surface systems.
matrix, by diffusion of the drug due the swelling of the polymer matrix n
and by the release due to degradation and erosion of the matrix [45]. As k. t
Q/ Q = 1 1
swelling of the matrix is meant the occurrence of three subsequent C0. a0 (8)
steps: entry of water molecules into the hydrogel, relaxation of the
k is the erosion rate constant, a0 is the initial radius for a cylinder or
polymer chains and stretching of the entire chains in the water [46]. To
sphere, and C0 is the initial drug loading. The n exponent varies with
evaluate the occurrence of such mechanisms and to predict drug release
geometry (2 for cylinder and 3 for sphere). According to this model, the
rates, several mathematical models can be found in the literature
limiting step of the release is the erosion of the matrix. In addition, the
[47,48].
internal and external diffusion resistances do not influence it.
Zero-order model (Eq. (3))
It is a kinetic model, which considers that the release of drug from
2.4.4. Analytical characterization techniques
the polymer matrix does not depend on its concentration. According to
Particles were characterized for crystallinity (XRD, X'Pert-MPD X-
this model, the same quantity of drug is released per unit of time over
ray diffractor, Philips, Cu Kα radiation, 40 kV, 40 mA, step size 0.02,
all time, this is ideal for extended release systems.
scan rate 0.02 s−1, range: 5–50°); morphology (SEM, Leo 440i Scanning
Q = k 0. t (3) Electron Microscope, Oxford, 200 Å gold-coated particles); functional
groups (FTIR, Nicolet 6700 Infrared Spectrometer, Thermo Scientific,
Q is the release of drug at time t, and k0 is the zero order release
transmittance mode, KBr method, range: 4000–400 cm−1, resolution:
constant.
4 cm−1); size and size distribution (OM, DC4-456H Stereo Microscope,
First-order model (Eq. (4))
National) and thermal stability (TG/DTG and DTA, DTG-60 thermal
In this case, the rate of drug release is concentration dependent. For
analyzer, Shimadzu, N2 flow rate: 50 mL min−1, heating rate:
this reason, the amount of drug released tends to decrease with time.
20 °C min−1, range: 30–1000 °C).
ln Q = ln Q0 k1. t (4)

where k1 represents the first order release constant. 3. Results and discussion
Higuchi model (Eq. (5))
Higuchi proposed one of the most famous and most widely used 3.1. Naproxen particles: drug loading capacity and entrapment efficiency
models to explain the drug release from solid/semi-solid matrix sys­
tems. This theoretical model (Eq. (5)) considers Fickian diffusion as the In Fig. 1 it is presented the results of EE and DL for the different
main process for drug release. formulations developed herein, besides images of the particles ob­
tained.
Q = kH . t 1/2 (5) It is observed that all the formulations generated independent

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

Fig. 1. Entrapment efficiency, drug loading capacity and photographs of the particles obtained for the NPVA, NDSP, NPEG, NSER and NALG formulations.

particles and with well-defined format. Except for NPVA, all the others Katsikogianni and Avgoustakis [55] who evaluated three model drugs
had a shape near the spherical one. In the case of NPVA, an elongation encapsulated into PLGA-mPEG nanoparticles. In the case of the present
of the particles is observed, caused by PVA, which increased the solu­ study, the value of DL interferes with the amount of particles to be
tion viscosity and, consequently, made dripping difficult, contributing placed in gelatinous capsules. The lower the DL, the greater the amount
to the non-sphericity of the particles. This property of PVA is well of particles required to achieve the same drug dosage.
known and is even used in the industry to increase viscosity in oph­
thalmic products [52].
EE allows evaluating the amount of drug that has been effectively 3.2. In vitro drug release
incorporated, relative to the amount initially added. Low values of this
parameter indicate greater loss of raw material during the preparation The release in simulated gastric medium was evaluated to determine
of the particles. From Fig. 1, we can observe that all formulations the gastro-resistant characteristic of the different matrices developed
presented high values of EE. Through the Tukey test, only the NALG herein. Both the alginate and the sericin/alginate matrices had their
formulation, with the lowest EE (73.07 ± 4.13%), presented a statis­ gastro-resistance proven in previous studies [18]. However, gastric
tically different result from the others, with 95% confidence. Thus, media assays were performed to confirm this property in all of the
NPVA, NDSP, NPEG and NSER are statistically equivalent, with effi­ formulations proposed herein. Table 1 presents the obtained results for
ciencies above 80%. the five formulations.
This result evidences the fundamental role of sericin in enhancing According to U.S. Pharmacopoeia [44], acidic drug releases of less
the capacity of entrapment of naproxen. None of the crosslinking agents than 10% characterize delayed (or gastro-resistant) dosage forms. Thus,
evaluated were able to improve the result already obtained only by the results in Table 1 indicate that only the NDSP formulation did not
adding sericin to the alginate. This may have occurred because of the show the expected gastro-resistance, showing that this formulation was
presence of the strong polar side groups in the sericin composition, not capable of protecting the drug from the acid medium action. All
which are capable of strongly crosslinking the alginate, avoiding the other formulations had their delayed release confirmed.
loss of drug throughout the process of particle production. In the for­ The release in simulated enteric media (pH 7.4) generated the dis­
mulation containing only alginate, this phenomenon is not observed, solution curves of Fig. 2.
allowing drug crystals to be lost before its complete incorporation [53]. We can observe that the formulations presented different behaviors.
DL is the mass ratio of drug to particles, indicating the percentage of NPVA presented a prolonged release character (~360 min). However, it
mass of particles that is due to the entrapped drug, and is affected by
the structure and physicochemical properties of the polymer matrix Table 1
[54]. From Fig. 1, DL varied in the range of 18.14 ± 0.67% to Release results in simulated gastric media (pH 1.2), obtained
30.44 ± 1.72%. In the case of maximum DL achieved, this value for the formulations NPVA, NDSP, NPEG, NSER and NALG,
means that there are about 304 mg of drug in 1 g of particles. According after 2 h of test.
to the Tukey test, with 95% confidence, the following pairs, NPVA and Formulation Acidic drug release (%)
NDSP, NPEG and NSER are statistically equal. The NALG formulation
NPVA 6.8 ± 1.4
had the highest DL capacity and was statistically different from all
NDSP 11.1 ± 1.8
others. NPEG 7.5 ± 0.5
According to the previous results, DL increased as the proportion of NSER 8.1 ± 1.4
drug in the formulation also increased. Similar result was observed by NALG 8.5 ± 1.3

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

This result was due to interference of the sericin/alginate/PEG particle

(blank particle), which exhibits high absorbance at the wavelength of
naproxen, preventing results related to the drug alone.
NALG presented prolonged release (~300 min), but reached its
equilibrium after only 90% of the drug release. This means that alginate
plays a key role in prolonging the release of naproxen, but it still pre­
vents the release of the entire drug present. The best formulation,
therefore, is NSER (sericin/alginate/naproxen), which reached a release
of around 98% between 300 and 360 min. Such formulation, with high
EE and DL, and gastro-resistant behavior, is the one with the best re­
lease profile. In addition, it becomes interesting in the face of NPVA,
NDSP and NPEG, as it does not require the presence of a covalent
crosslinking agent to achieve the expected result.
The result of the present work was better than that obtained by
Čalija et al. [38] who evaluated the release of naproxen from alginate/
oligochitosan/Eudragit® L100-55 microparticles. According to their
results, the maximum drug release time in simulated enteric media
(pH 7.4) was 2 h. The results reported by Dyakonov et al. [56] were
Fig. 2. Drug release curves in simulated enteric media (pH 7.4) for NPVA, very close to the results published here. The release of naproxen from a
NDSP, NPEG, NSER and NALG formulations. The error bars correspond to the sericin/gelatin/glycerin blend took about 350 min to equilibrate.
standard deviation obtained for the tests performed in triplicate. However, unlike the present study, those authors achieved only 80%
release at equilibrium. Thus, the importance of the sericin/alginate
reached equilibrium value greater than 100%, indicating that the hy­ blend is reached to promote a better release of naproxen.
pothesis assumed in the theoretical calculation of the EE may not have
been satisfied for this formulation. It is possible that raw material was 3.3. Mathematical modeling of drug release
lost during the preparation of particles, leading to errors in determining
the EE and consequently the amount of particles employed in the dis­ The parameters of the adjusted models, as well as the Akaike
solution test. Information Criterion (AIC) and the adjusted coefficient of determina­
NDSP showed near zero release until 120 min and then had a rapid tion (R2adj) are presented in Table 2. The use of the R2adj occurred because
release reaching equilibrium in 240 min. Such behavior is not inter­ it eliminates the limitation of the coefficient of determination R2, which
esting, because the drug keeps trapped in the polymer matrix and, only decreases if an explanatory parameter is taken from the model,
therefore, has its therapeutic action delayed. In addition, this is the only regardless of its relevance [57]. The AIC is a useful tool, especially for
non-gastro-resistant formulation among those obtained. In the case of models with different number of parameters comparison, including
NPEG, its equilibrium reached only 63% of removal, in about 300 min. incompatible models [58].
Zero-order and first-order models are useful to provide details on

Table 2
Parameters obtained from the adjustment of the mathematical models of zero and first order, Higuchi, Weibull, Korsmeyer-
Peppas and Hopfenberg for cylindrical (n = 2) and spherical (n = 3) systems, to the experimental data of drug release in
simulated enteric medium. To assess the best adjustments, the values of the adjusted coefficient of determination (R2adj) and the
Akaike Information Criterion (AIC) are presented. The model that best fit each formulation is represented by the emphasis in
bold and italic to the value of R2adj.
Model Parameter Formulation

NPVA NDSP NPEG NSER NALG

Zero order k0 0.323 0.325 0.215 0.370 0.338

R2adj 0.855 0.500 0.837 0.860 0.933
AIC 89.40 81.27 69.63 79.48 68.71
First order k1 0.006 0.004 0.003 0.006 0.006
R2adj 0.778 0.264 0.715 0.629 0.853
AIC 90.66 85.10 74.9 95.05 74.46
Higuchi kH 4.84 3.40 2.75 4.84 4.54
R2adj 0.528 0.097 0.295 0.371 0.524
AIC 100.35 89.26 85.89 95.05 87.48
Weibull a 12,849 12,588 28,753 434,683 2150
b 1.905 1.867 1.833 2.620 1.510
Td 144 157 271 142 161
R2adj 0.977 0.609 0.942 0.996 0.981
AIC 68.02 82.05 60.61 42.51 55.93
Korsmeyer-Peppas kKP 0.005 1.00⁎10−9 0.003 0.001 0.108
n 2.29 4.79 1.79 2.35 1.27
R2adj 0.993 0.933 0.958 0.978 0.916
AIC 21.11 44.21 47.63 37.81 49.41
Hopfenberg n = 2 k2 0.003 0.002 0.001 0.003 0.002
R2adj 0.885 0.361 0.785 0.785 0.934
AIC 84.33 83.55 72.22 82.54 66.89
Hopfenberg n = 3 k3 0.002 0.001 0.001 0.002 0.002
R2adj 0.857 0.324 0.763 0.736 0.914
AIC 86.31 84.12 73.14 84.45 69.42

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

Fig. 3. (A) X-ray diffraction patterns obtained from the pure naproxen (NPX), from the NPVA, NDSP, NPEG, NSER and NALG formulations and their respective non-
drug particles (BPVA, BDSP, BPEG, BSER and BALG). (B) X-ray diffraction patterns of pure naproxen (NPX) and drug-loaded formulations (NPVA, NDSP, NPEG, NSER
and NALG) showing the intensity of the diffraction peaks.

the kinetics of drug release, while the other models provide details 3.4. Analytical characterization
about the mechanism under which such release occurs [47]. From
Table 2, regardless of the formulation, the worst adjustments occurred 3.4.1. Cristallinity
for the first-order and Higuchi models. This means that the drug release In Fig. 3(A), the diffractograms obtained for drug particles, for the
kinetics is independent of its concentration in the polymer matrix and non- drug particles, identified by the initial letter B, and for the pure
also does not follow the diffusion mechanism, assumed by the Higuchi drug (NPX) are presented. On Fig. 3(B), it is highlighted the diffracto­
model [59]. grams of the five formulations and the NPX considering their peak in­
The NDSP formulation, which presented a very different behavior tensities.
from the others and had its drug release profile approaching a step The diffractograms of BPVA, BPEG, BSER and BALG have an
function, only presented a good adjustment to the Korsmeyer-Peppas amorphous characteristic, with broad peaks representative of the
model. None of the other models predicted the data obtained in this components of the particle. Sericin typically presents a peak close to
case, with low R2adj values. The NPVA, NPEG and NSER formulations 20.5° due its β-sheet conformation [63]. Alginate has peaks at 13.5°, 22°
had the best fit for the Korsmeyer-Peppas and Weibull models. and 39°, related to polyguluronate, polymannuronate and amorphous
Regarding the Korsmeyer-Peppas, all presented values of n > 0.89, halo structures, respectively [64]. In Fig. 3(A), we observe the presence
including NDSP and NALG, indicating Super Case-II transport. Thus, the of all these peaks, with emphasis on the broad peak near 22°, as a
swelling and relaxation of the polymer chains of the blend are re­ function of the interaction between sericin and alginate. BDSP shows
sponsible for the release of the drug in all cases [60]. Papadopoulou amorphous structure with pronounced peaks at 25° and 30°, due to the
et al. [61] confirmed the relationship between the adjustment of the presence of the crystalline DSP interacting with the amorphous sericin/
Weibull model and the prediction of the mechanism associated to the alginate blend [65].
release of drugs. Values of parameter b > 1.0 for all formulations in­ In the NPX diffractogram, the crystalline nature of the drug is ob­
dicate complex release mechanism with the dissolution profile as a served, with sharp and distinct peaks (2θ = 6.62°, 12.65°, 16.76°,
sigmoid shape. A complex release mechanism is the occurrence of dif­ 18.97°, 20.33°, 22.58°, 23.68° and 28.35°). All of these peaks were ex­
ferent mechanisms, such as erosion, diffusion and swelling of the ma­ pected for naproxen, as previously reported [66,67]. In the drug par­
trix, confirming the result obtained by the previous model adjustment ticles diffractograms, the major peaks of naproxen can still be observed,
[62]. but there is a shift to a more amorphous structure. This reduction in
Almost all models (zero-order, Korsmeyer-Peppas, Weibull and crystallinity means that a physical mixture or dissolution of naproxen in
Hopfenberg) adjusted well the data of the NALG formulation. This the polymer matrix occurred in all cases, confirming the drug in­
means that release kinetics was independent of concentration and that corporation. In Fig. 3(B) the diffractograms of the five formulations and
the release mechanism was a mixture of more than one phenomenon, the NPX are highlighted, in order to prove the reduction of crystallinity,
including erosion, swelling and relaxation of the polymer chains. by the reduction in the peaks intensity. We observe that the reduction
was quite significant and that in the case of NALG, there was the
greatest change in the structure of the drug, for an amorphous

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

Fig. 4. Micrographs obtained by SEM from pure naproxen (NPX), non-drug formulations (BPVA, BDSP, BPEG, BSER and BALG) and drug formulations (NPVA, NDSP,
NPEG, NSER and NALG), at (a) 3000× magnification and at (b) 150× magnification. The micrographs of the particles were obtained (a) from the cross-section of the
inner surface and (b) from the outer surface.

characteristic. The shift to the amorphous form directly affects solubi­ 3.4.2. Morphology
lity, since these structures are more soluble than organized crystalline Through SEM technique, micrographs of non-drug particles and
structures. This is interesting because the objective is to obtain phar­ drug particles were obtained in addition to the pure drug itself. The
maceutical forms of gradual release, thus increasing the solubility fa­ micrographs with magnification of 3000× were obtained from the in­
vors release by swelling the polymer matrix [23]. ternal surface of the particles, through a cut in the cross-section. Fig. 4
shows these micrographs.

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

Fig. 5. Infrared spectra obtained by FTIR presented in Transmittance mode of (A) non-drug particles (BPVA, BDSP, BPEG, BSER and BALG) and of (B) pure naproxen
(NPX) and drug particles (NPVA, NDSP, NPEG, NSER and NALG).

The non-drug particles presented different outer surface character­ 3.4.3. Functional groups
istics. BPVA presented depressions on its surface, as result of some voids In order to evaluate the changes of the blends by the addition or not
observed inside it. These voids indicate a particle with high porosity of covalent crosslinking agents, the spectra obtained for non-drug
and are the result of the presence of PVA, which alone is capable of particles are shown in Fig. 5(A). The spectra of drug and drug particles
forming a three-dimensional gel with high porosity, as reported by Neo are presented in Fig. 5(B).
et al. [68], who also observed high porosity in silk/PVA gels. BDSP had Despite the different intensities, all the spectra of Fig. 5(A) are quite
a very particular surface, with the presence of a rough porous structure similar. Since sericin is a protein, some characteristic bands of protein
not seen inside the particle. When the DSP binds to the blend, there is amides are observed: at 1630, 1540 and 1250 cm−1 referring to amide
the initial formation pre-polymers of lower molecular weight; with the I, amide II and amide III, respectively. The band referring to amide III is
crosslinking, the weight of this structure increases and allows the for­ the one with the lowest intensity and, in some cases, practically dis­
mation of particles. The pre-polymers, which did not completely appears. Possibly, there was interaction between the components of the
crosslink, deposit on the surface of the particles, making it more rough particles through this functional group. Regarding the sericin secondary
[69]. structure, bands at 1655, 1630 and 1645 cm−1 are equivalent to the α-
BPEG, BSER and BALG presented similar surfaces, with some cracks helix, β-sheet and random coil structures, respectively. In Fig. 5(A), the
present, and in the case of BPEG, with the presence of small surface position of this band varies in the range of 1635–1650 cm−1 for the
holes. BALG was the one that presented the most cracks, appearing to BPVA, BDSP, BPEG and BSER particles [70]. This indicates that there is
be the most fragile. Internally, all the formulations had similar surface, a tendency for the organized structure of β-sheet (insoluble) to change
presenting a fairly smooth and homogeneous surface. Only in BDSP and to amorphous structures of random coil (soluble), which facilitates
BPEG there is a small formation of layers, possibly resulting from the swelling and dissolution during drug release. An insoluble particle
presence of the crosslinking agents. would not be applicable to the pharmaceutical field since it would
Comparing NPVA, NDSP, NPEG, NSER and NALG, external surfaces prevent the drug from leaving.
are very similar, with a rough characteristic due to the entrapment of Alginate also exhibits some characteristic bands, e.g. in the range of
the drug in the blend. In NPVA, the non-spherical shape of the particle 1000–1200 cm−1 by the C – O absorption of the saccharide of poly­
is remarkable, as was already observed. The high viscosity of the so­ saccharides and at 1427 cm−1, as a function of the C = O symmetrical
lution contributed to this shape, preventing well-defined spheres from stretching of the carboxylic acid salts [71]. There are also alginate
forming during the dripping process. In the case of NDSP, the outer bands at 3440 cm−1 (hydroxyl groups), 824 cm−1 (mannuronic acid),
surface was significantly altered by the presence of the drug, which 894 and 943 cm−1 (guluronic acid) [72]. All of them appear in all of
made the crosslinking process more homogeneous. The inner surface of the samples of Fig. 5(A). PVA bands in BPVA (2986 cm−1 of the C – H
the drug particles (identified by subscript (a)) indicate the presence of stretching and 1081 cm−1 of the C – O stretching) and PEG bands in
the drug incorporated in all cases. In order to confirm this incorpora­ BPEG (at 1100 cm−1 of the C – O stretching) can also be identified
tion, the analysis of FTIR (presented below) allows verifying the func­ [21,73,74].
tional groups of the formulations. In the NPX spectrum of Fig. 5(B), it is possible to identify char­
acteristic bands of the functional groups of the drug: at 3188 cm−1 (O –

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

Fig. 6. Particle size distributions obtained from the NPVA, NDSP, NPEG, NSER and NALG formulations, presented in the form of relative frequency (□) and
cumulative frequency ( ), in addition to the Gauss fitting (normal) ( ) to the relative frequency of the distribution. In each profile, the mean diameter ( ) obtained
for each formulation and the respective adjusted determination coefficient ( ) of the Gaussian fitting are also highlighted in red. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

H), at 3002 cm−1 (O – CH3), at 2963 cm−1 (CH3) and at 1728 cm−1 (C was determined using ImageJ™ software. Thus, the size distribution and
= O) [75]. Similar bands are observed in the spectra of all formulations mean particle diameter (d) were obtained, and are presented in Fig. 6.
developed, indicating that the drug was entrapped by the polymer In the Figure it is also shown the values of R2adj of the Gauss fitting
blend, without interacting with its components. Except for the band at applied to the Relative Frequency.
1728 cm−1, all the others shifted to greater values, possibly because The Tukey test of the mean diameter data of Fig. 6 indicated that,
they also represent functional groups of the polymer matrix. The hy­ statistically, with 95% confidence, all diameters obtained were
droxyl group, for example, is present in the drug, but also in the algi­ equivalent. Thus, even the addition of crosslinking agents did not in­
nate and water that may be present in the particles. The C = O func­ terfere with particle size. The obtained size range is suitable for dosage
tional group may also be present in both the drug and the alginate. forms of granules in oral administration system [76]. The size dis­
Thus, the spectra confirm the incorporation of the drug and indicate tribution and the R2adj values indicated that, in general, all formulations
that no major changes occur as a function of the presence or absence of had a homogeneous size distribution, with the worst case for NPVA
covalent crosslinking agents. (R2adj = 0.902), which was expected, since it did not present a spherical
shape. In addition, except for NPVA, all have a narrow size distribution.
3.4.4. Size/size distribution
Particles images were captured through OM technique at 10× 3.4.5. Thermal analyzes
magnification, and the diameter of 500 particles of each formulation TG/DTG and DTA were performed on pure drug (NPX) and the drug

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E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

Fig. 7. Curves obtained from thermogravimetry analysis (TG) given in Weight loss (–) and its derivative (DTG) ( ) and from differential thermal analysis (DTA) ( )
for pure naproxen (NPX) and for the NPVA, NDSP, NPEG, NSER and NALG formulations. The TG derivative was obtained as a function of time, but represented by the
increase in temperature.

particles. The results obtained are shown in Fig. 7. 170–370 °C due the destruction and degradation of glycosidic bonds,
For better visualization, in the curves of NPX the y-axis to the left, which contributes to the mass losses of the obtained formulations in this
equivalent to TG/DTG, was divided in two. The TG/DTG curves in­ temperature [79]. PEG degrades in a single stage of mass loss, close to
dicate that the drug is stable up to 200 °C and has a single stage of mass 400 °C. Thus, in NPEG, there is a small loss of mass at this temperature
loss between 200 and 300 °C. The DTA curve shows two endothermic [80].
events: close to 160 °C, relative to the melting point of the material, and The DTA curves of all formulations present similar events, but with
close to 300 °C relative to its decomposition and related to its mass loss different intensities. The endothermic events are also related to drug
in TG/DTG. This behavior was expected [77]. melting (~160 °C) and polymer matrix decomposition (~200 °C). In
The mass loss curves of the five formulations show very similar case of alginate, in the range of 202.8–585 °C, it usually presents an
behavior, with mass losses occurring in more than one stage, especially initial dehydration followed by degradation and formation of Na2CO3
between 200 and 300 °C. This may be related to drug decomposition, and carbonized material. Thus, the exothermic peak near 600 °C, ob­
but also to the effect of temperature on the polymer matrix. Sericin and served in some of the DTA curves of Fig. 7 and quite marked in NALG
PVA decompose in the range of 250–300 °C and therefore the peaks of curve, is equivalent to this decomposition [81].
the DTG curve of NPVA, at 200 and 300 °C, are not as independent [78]. Despite the drug-related events, it is observed that its mass loss is
The alginate exhibits a characteristic mass loss in the range of much more significant than when incorporated into the blend. Thus, we

10
E.D. Freitas, et al. Materials Science & Engineering C 118 (2021) 111412

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Melissa G. A. Vieira: Funding acquisition; Project administration;
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its pharmacological properties and therapeutic efficacy and use in rheumatic dis­
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[21] E.K. Solak, Preparation and characterization of IPN microspheres for controlled
delivery of naproxen, J. Biomater. Nanobiotechnol. 02 (2011) 445–453, https://
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[22] E.D. Freitas, P.C.P. Rosa, M.G.C. da Silva, M.G.A. Vieira, Development of sericin/
alginate beads of ketoprofen using experimental design: formulation and in vitro
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Technological Development, CNPq (Grant numbers 470615/2013-3, 1016/[Link].2018.05.016.
301401/2016-0) and The São Paulo Research Foundation, FAPESP [23] E.D. Freitas, B.M. Lima, P.C.P. Rosa, M.G.C. da Silva, M.G.A. Vieira, Evaluation of
proanthocyanidin-crosslinked sericin/alginate blend for ketoprofen extended re­
(Grant numbers 2015/13505-9, 2016/05007-1). The authors are lease, Adv. Powder Technol. 30 (2019) 1531–1543, [Link]
grateful for Bratac Silk Mills Company for the supplying of silkworm 2019.04.031.
cocoons. [24] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and in­
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