International Journal of Pharmaceutics 609 (2021) 121184

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Computational analysis of vincristine loaded silk fibroin hydrogel for

sustained drug delivery applications: Multiphysics modeling
and experiments
Ziba Gharehnazifam a, Roshanak Dolatabadi b, Majid Baniassadi a, Hamid Shahsavari a,
Abdol-Mohammad Kajbafzadeh c, *, Karen Abrinia a, Mostafa Baghani a, *
a
School of Mechanical Engineering, College of Engineering, University of Tehran, Tehran, Iran
b
Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
c
Pediatric Urology and Regenerative Medicine Research Center, Children’s Medical Center, Pediatric Center of Excellence, Tehran University of Medical Sciences, No. 62,
Dr. Qarib St, Keshavarz Blvd, 4194 33151 Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: In this paper, silk fibroin hydrogel is used as a drug carrier for vincristine. To optimize drug delivery, a multi-
Silk fibroin hydrogel physics model is proposed that couples the deformation and diffusion fields. We applied inverse analysis and
Sustained drug delivery general continuum mechanics to define material parameters and mechanical properties. To examine the mass
Multiphysics
transport and chemical behavior, an affinity-based diffusion and degradation of a drug-loaded polymer matrix is
Affinity based diffusion
Hydrogel degradation
employed. Some experiments are carried out to examine the capability of the presented model. After preparing
Hydrogel Swelling the vincristine loaded silk hydrogel syringes, they were injected into PBS and enzyme solutions to monitor the
drug release rate for 40 days. Obtained results from the computational simulation and laboratory tests showed
that the silk fibroin hydrogel was deswelled after about 40 days in enzyme solution. Degradation led to faster and
higher doses of vincristine drug release in comparison to the case of PBS solution. Results revealed that more
than 80% of the drug was released in the first 5 days in the enzyme solution, but in PBS solution only 10% of the
drug was released during 40 days. The model predictions of deswelling behavior and drug release rate were in
good agreement with those of experimental results. Therefore, it can be employed as a reliable tool for further
predictions.

1. Introduction
According to the report of the world health organization, cancer is
the second main reason of death in the world, that included one sixth/
9.6 million deaths in 2018 (WHO, 2018). Globally, it has the most
harmful economic impact among other causes of death. In 2010, its total
annual treating costs were approximately US$ 1.16 trillion (Wild et al.,
2014). This indicates the severity and negative effects of cancer on
human society. Therefore, research on the most optimal treatment
methods is of great importance. Targeted drug delivery has been one of
the most favorite treatment methods in recent years.
Targeted drug delivery also known as smart drug delivery, is an
approach for delivering the therapeutic material to the targeted site so
that it does not cause undesirable effects in other organs. There are
several advantages to this method compared to conventional drug
delivery, including reduced toxic side effects, reduced cost, reduced dose
and improved efficacy. This makes it effective against cancers such as
those of the lung, the breast, the kidney, the bladder and the prostate
(Kundu et al., 2019; Baghani et al., 2017; Afrasiabi Garekani et al.,
2017). Targeted drug delivery can be accomplished through hydrogels,
nanoparticles, carbon nanotubes, micelles, liposomes and microfluidics
(Iqbal et al., 2017). Hydrogels are an attractive choice due to facilitating
a controlled release of the drug (Narayanaswamy and Torchilin, 2019).
Encapsulating hydrophobic drugs, such as vincristine and doxorubicin
which are mainly used for treatment of the cancers listed above, is a real
challenge that will be addressed in this study (Larrañeta et al., 2018).
Silk fibroin, isolated from the cocoon of silkworm Bombyx mory, is a
high molecular weight polymer which contains two major parts, light
(~25 KDa) and heavy (~325 KDa) fibroins (Numata and Kaplan, 2010;
Tamada, 2005). In the context of drug delivery and sustained drug

* Corresponding authors.
E-mail addresses: kajbafzd@tums.ac.ir (A.-M. Kajbafzadeh), baghani@ut.ac.ir (M. Baghani).

https://doi.org/10.1016/j.ijpharm.2021.121184
Received 9 July 2021; Received in revised form 5 October 2021; Accepted 8 October 2021
Available online 12 October 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.
Z. Gharehnazifam et al.
Fig. 1. Hydrogel drug delivery investigation methods.
release, this material is highly attractive due to its excellent bio-
degradability, bio-compatibility, tunable mechanical properties, and
ability to form a crystalline hydrophobic matrix (Numata et al., 2010;
Meinel et al., 2004; Horan et al., 2009). Silk fibroin can be employed in
different forms of hydrogels (Ribeiro et al., 2018); films (Hines and
Kaplan, 2011); fibers (Farokhi et al., 2020), nanoparticles (Song et al.,
2017), nano/microspheres (Wang et al., 2007; Wang et al., 2010) and
sponges (Pritchard et al., 2013) in drug delivery applications. Silk
fibroin hydrogels can be used to encapsulate hydrophobic drugs, due to
their β-sheet structure (Elliott et al., 2015).
Generally, hydrogel drug delivery has been explored through release
rate monitoring, swelling studies, in vitro degradation studies, in vitro
cytotoxicity studies, in vivo tumor studies and analytical methods as
depicted in Fig. 1 (Vigata et al., 2020; Seib et al., 2013). Seib et al.
(2013); Harris et al. (2016) and Wu et al. (2016) experimentally
analyzed the release behavior of silk fibroin hydrogel. They utilized
vincristine or doxorubicin as a therapeutic material and showed that the
silk fibroin hydrogel is a good candidate for sustained drug release. In
order to maximize the control parameters, mathematical simulations are
a favorite tool because experiments are costly and time-consuming
(Vigata et al., 2020).
In order to arrive at optimal drug delivery results, mathematical
simulation is one of the best approaches. In this regard, a comprehensive
knowledge of the drug release physics and hydrogel material charac­
teristics is required. The main mechanisms for sustained drug release in
this material include diffusion, chemically release controlling, swelling
and combinations of diffusion with degradation or swelling. Among
these phenomena, diffusion plays the most important role which is most
frequently used to model the drug delivery behavior in hydrogels. Drug
diffusion can be simulated employing different models including free
volume, hydrodynamic, obstruction and combination of hydrodynamic
and obstruction (Hines and Kaplan, 2011; Yucel et al., 2014; Pritchard
et al., 2010; Uebersax et al., 2008; Bessa et al., 2010). Swelling-
controlled release model can be applied when the rate of drug release
is faster than the hydrogel swelling (Siepmann and Peppas, 2012;
Dolatabadi et al., 2021a; Bayat et al., 2020a; Arbabi et al., 2017; Sho­
jaeifard et al., 2021). In chemically controlled release mechanisms, all
2
International Journal of Pharmaceutics 609 (2021) 121184
reactions related to the drug loaded polymer matrix such as degradation
of polymer matrix and affinity-based bindings of drug and polymer
should be taken into account. In this mechanism, surface or bulk
degradation controls the drug release rate. Affinity-based equations are
utilized to account for the binding reactions between the drug and
polymer matrix (Peppas et al., 2000; Amsden, 1998; Kanjickal and
Lopina, 2004).
As mentioned before, diffusion is the most common model used for
this type of hydrogel. Thus, some mathematical models, such as zero
order, first order, Higuchi, Peppas models and Fickian diffusion, have
been employed. Peppas power law is the most frequently applied
diffusion model for hydrogel drug delivery (Peppas et al., 2000; Ritger
and Peppas, 1987). Hines and Kaplan (2011) compared the Fickian
diffusion model with the empirical power law one to examine the effect
of various parameters on the release rate, such as lowering molecular
weight and methanol treatment. Other complex mechanisms such as
degradation, swelling and chemical interactions can be added to this
diffusion model to have a more accurate prediction (Lin and Metters,
2006; Siepmann and Siepmann, 2008; Siepmann and Peppas, 2001;
Artifin et al., 2006; Chilin and Metters, 2006).
Usually, a constant release of the drug cannot be happened. There­
fore, another mechanism such as degradation is needed to be combined
with the diffusion (Hines and Kaplan, 2011). Degradation in silk mate­
rials is heterogeneous (surface degradation) when it is immersed in an
enzyme solution. In heterogeneous materials we have zero order release
(Peppas et al., 2000). In this kind of matrix, binding between the drug
and matrix is very significant and can be engineered to possess a suitable
drug release. Wongpanit et al. (2010) examined two types of solvent to
observe the release from silk fibroin scaffolds, enzyme solution and
water. As a result of a higher rate of degradation, the enzyme-infused
medium enhanced the release rate. In silk fibroin, degradation rate
can be controlled by lowering the molecular weight through applying a
longer degumming time (Pritchard et al., 2013) or making compositions
with other polymers (Mandal et al., 2009a, 2009b; Zhong et al., 2015).
Diffusion can be affected by mechanical properties of the material; in
parallel, the mechanical behavior of the material is highly influenced by
degradation phenomena, as well. These dependencies can be taken into
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

Fig. 2. Experimental and finite element simulation steps.

Fig. 3. (A) Cocoons of Bombyx mori silk, (b) cut pieces of cocoons of Bombyx mori silk, (c) silk fibers under a microscope, d: Scanning electron microscopy (SEM)
image of silk fibroin solution after lyophilizing.

control is taken into account in this type of diffusion, it is commonly

named Non-Fickian case II diffusion. Several attempts have been con­
ducted to present coupled modeling of mechanical and diffusion fields to
observe their effect on the swelling behavior. Non-Fickian case II
diffusion models have usually been used in these researches (Liu et al.,
2016; Hong et al., 2008; Bargmann et al., 2011).
In this study, we investigated the release of vincristine from silk
fibroin hydrogel to further develop targeted drug delivery for the
treatment of Wilm’s tumor (a type of kidney cancer seen in children).
Treatment with vincristine is common for this type of cancer. In this
paper, vincristine loaded silk fibroin hydrogels were prepared and
injected into the Phosphate-buffered saline (PBS) and Type II Collage­
nase enzyme solutions to monitor the release behavior of hydrogel
during 40 days. Swelling test was performed by weighting the hydrogel
at different times to observe its deswelling response. Moreover, a
rheological test was carried out to measure mechanical properties of the
material such as loss and storage moduli. To model the drug release from
silk fibroin hydrogel, a combination of diffusion and chemically
controlled mechanism was conducted. A coupled model of mechanical
behavior and diffusion was applied to have an accurate prediction of the
drug release. In addition to diffusion, the effect of chemical reactions
including the surface degradation of silk fibroin matrix by enzyme, and
the binding reactions between the vincristine drug and matrix, was
considered. In numerical simulations, three interfaces of COMSOL-
Multiphysics software, i.e., diffusion of diluted species, chemical re­
actions and solid mechanics were coupled. Affinity based diffusion
equations and Michaelis-Menten kinetics were used to properly model
Fig. 4. A syringe containing vincristine loaded hydrogel that is incu­
the diffusion and degradation in the vincristine loaded silk fibroin
bated overnight.
hydrogel. Rheometry results were employed to callibrate material pa­
rameters of the finite strain hyperelastic constitutive model. Finally, the
account by employing a coupled model. Hence, methods that couple
simulation results were compared to the results obtained from the ex­
diffusion and deformation can be utilized to have a more realistic pre­
periments to confirm the accuracy of the presented simulations. Based
diction of the hydrogel behavior and its effect on drug delivery appli­
on the given description, Fig. 2 shows the steps of the research taken in
cations. In contrast to classical diffusion, Non-Fickian diffusion is
this paper.
coupled with other physical and physical–chemical processes. In this
case, diffusion is affected by relaxation of molecules, segmental mobility
retardation, functional interactions between molecules, crystallization
and other factors. Additionally, in terms of Non-Fickian diffusion, sur­
face or volume anomalous processes could be identified. If swelling

3
Z. Gharehnazifam et al.
Fig. 5. Silk fibroin hydrogels in PBS and enzyme solutions.
2. Materials and experiments
2.1. Materials
In this study, silk fibroin hydrogel was used as a drug carrier and
vincristine as a therapeutic material. Vincristine was purchased from
Sobhan-oncology group. Bombyx mori silk fibroin cocoons were sup­
plied from the northern part of Iran (Darvazehmelal Company). Type II
collagenase enzyme was purchased from Merck group.
2.2. Fabrication of silk fibroin hydrogel
According to DN Rockwood protocol (Rockwood et al., 2011), after
cutting cocoons of Bombyx mori silk, they were boiled in a 0.02 M so­
lution of Na2CO3 for 30 min. Following overnight drying, 9.3 M LiBr was
added on top of the fibers and they were incubated at 60℃ for 4 h. The
remaining solution, were dialyzed against distilled water for removing
the salts (48 h). The solution was centrifuged twice and then stored at
4℃. According to this protocol, concentration of silk fibroin solution
should be about 7–8 wt% (Fig. 3). Sonicating the silk fibroin solution for
5 s and incubating it in an incubator for 24 h are all that is needed to
prepare the hydrogel.
2.3. Vincristine loaded hydrogel preparation
After sonication of the silk fibroin solution for 5 s, a certain amount
of vincristine was added to the solution. For 100 μL of silk fibroin so­
lution with concentration of 0.6% (w/v), 50 μg of vincristine was added
(Harris et al., 2016). Then, the mixture of drug and silk fibroin solution
was aspirated into syringes and allowed the gel to stay inside the incu­
bator overnight, see Fig. 4.
2.4. In vitro drug release study
Following the formation of the drug-loaded hydrogel, the syringes of
the hydrogel were injected into a 24 well plate which contained 2 ml of
PBS or a mixture of PBS and enzyme, and was then placed in an incu­
bator. After one hour, the contents of the wells were removed and
replaced with PBS or PBS mixture with enzyme (see Fig. 5). This pro­
cedure was repeated on days 1, 5, 12, 19, 26, 33, and 40. These time
periods were chosen to monitor the release behavior during 40 days.
Drug concentrations of the removed samples were measured by
UV–Visible at the wavelength of 298 nm (Sun et al., 2016).
2.5. Hydrogel deswelling
After gelation of 200μL silk fibroin hydrogels, they were weighed and
transferred to some cells with PBS and some cells with type II collage­
nase enzyme reconstituted in PBS, to study the deswelling effect.
4
International Journal of Pharmaceutics 609 (2021) 121184
Weights of these two sets of hydrogels were measured on days 1, 5, 12,
19, 26, 33, and 40. After each measurement, PBS and type II collagenase
enzyme solution were recharged in each of the cells. Deswelling ratio
can be determined through (Singh et al., 2009):
Weight(t > 0)
Qm = . (1)
Weight(t = 0)
2.6. Rheometery test
Rheometery tests are required to determine the mechanical proper­
ties of hydrogels, including their modulus of loss and storage. The Anton
Paar MCR 502 rheometer was used for this purpose. A silk fibroin
hydrogel with a diameter of 25 mm and a height of 2 mm was prepared
for the rheology test. The test was performed at a frequency of 1 Hz and
with a shear rate of 0 to 1%.
3. Mathematical modeling
To predict the release response of the silk fibroin hydrogel, a hybrid
mathematical model was developed. This model is a combination of an
affinity-based diffusion and degradation coupled with mechanical
behavior. In the following, the details of these mechanisms will be
discussed.
3.1. Mechanical modeling
The initial state of the hydrogel is swollen since the silk fibroin so­
lution has been mixed with the drug in earlier steps before being aspi­
rated into a syringe, and then allowed to swell. It is considered as a
nearly incompressible material that deswells over time.
In order to describe the polymeric gel behavior, Helmholtz free en­
ergy of the hydrogel due to stretching of the polymer, and mixing of the
polymer and small molecules as stated by Flory and Rehner (1943) is
employed. The following set of equations is used for calculating free
energy and obtaining volume fraction:
ψ (F, J) = ψ e (F) + ψ m (J)
∂2 u
ρ = ∇.(FS)T + Fv, F = I + ∇u (2)
∂t2
∂ψ 1
S = Sext +
∂ε
, σ = J − 1 FSFT , J = det(F), ε = (FT F − I)
2
where ψ e and ψ m stand for the free energy of stretching and mixing,
respectively; and F denotes the deformation gradient tensor. S is second
Piola-Kirchhoff stress, u is displacement and v is velocity. According to
Abi-Akl et al. (2019a), mixing free energy can be written as a function of
volume ratio J, which yields:
kT
[ (
1
)
χ
]}
ψ m (J) = (J − 1){ψ 0 + ln 1 − + (3)
η J J
where ψ 0 is the free energy of a unit volume of unmixed solvent, k ex­
presses the Boltzmann’s constant, T is the temperature,η indicates the
volume of a solvent unit, and χ denotes the dimensionless Flory–Huggins
interaction parameter.
Since the material exhibits a hyperelastic behavior, it can be modeled
through employing the Neo-Hookean strain energy. Thus, the following
general form is appropriate for this purpose (Holzapfel, 2002; Bayat
et al., 2020b):
( )
ψ e (F) = c1 I 1 − 3 + c2 (J − Js − 1)2 , (4)
where I1 is the first invariant of the left Green Cauchy deformation
tensor, B = FFT, and Js is the swelling ratio. A bar over a variable stands
for its deviatoric value.
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

where R is ideal gas constant and ξ is a parameter that relates the os­
motic pressure to the swelling ratio and can be found from the calibra­
tion. ck represents the concentration of the kth species, while c0k is the
concentration of the kth species in the surrounding medium.
A stored energy density function was used to calibrate material pa­
rameters. A linear regression analysis, as outlined in Appendix A (Abali
et al., 2019), and experimental measurements made using oscillatory
rheometry at a low frequency were employed to calibrate all strain en­
ergy material parameters. In rheometric measurements, the rate of the
stored energy and free energy are assumed to be equal. To determine the
material parameters, the energy can be recalculated via the following
equations.

Fig. 6. Binding interactions between species in affinity-based model. ∫T

e1 = ψ ∙ dt = 2π G˝ε20 (6)
0

Table 1
Variables/ constants used in mathematical modeling. ∫T/4
1
e2 = ψ ∙ dt − e1 = G’ε20 (7)
Constant Description Value 0 4
ψ0 Free energy of a unit volume of unmixed solvent − 4×105 J/m3
(Abi-Akl et al., 2019b) ε0 is strain amplitude, and e1 , and e2 represent the loss and storage en­
K Boltzmann’s constant (Brochure, 2019) 1.38 × 10− 23
J/k ergy densities, respectively. It should be noted that, G′′ (loss modulus)
T Temperature 298 k represents the dissipated energy and can be calculated from energy
H Volume of a solvent unit (Hong et al., 2008) 10− 28 m3 within the whole period, and G′ (storage modulus) represents the stored
χ Flory–Huggins interaction parameter (Hong 0.95 energy which can be found in a quarter period ignoring dissipated en­
et al., 2008; Whittaker et al., 2014) ergy (Abali et al., 2016).
R Ideal gas constant 8.3144 J/(K.mol)
ξ Osmotic parameter 1.916×10− 5 (1/Pa)
3.2. Affinity-based diffusion and degradation modeling
c1 Isochoric material parameter 1.5982 × 105 Pa
c2 Volumetric material parameter 1.1 × 106 Pa
A mathematical model was used to predict the drug release by
KM Michaelis-Menten constant (Wade et al., 2015) 3.09 mol/ m3
considering the matrix degradation and affinity between the matrix and
Vmax Maximum rate (Wade et al., 2015) 3.2 × 10− 9 mol/
(m3.s.unit)
the vincristine drug (Maxwell et al., 2005). Species in this model are free
kf Forward reaction rate constant, drug 0.5 × 106 m3/(s.
Vincristine (V), Bound Peptide (BP), Free Peptide (FP), Bound
dissociation (Chabner et al., 1978) mol) Vincristine-Peptide (BVP) and Free Vincristine-Peptide (FVP). Vincris­
kr Reverse reaction rate constant, drug dissociation 1 × 10− 5 1/s tine can associate and dissociate to free and bound peptides through
(Kar, 2003) electrostatic interactions. Kf and kr denote the forward and reverse in­
Cenzyme Initial concentration enzyme, biomaterial matrix 8 × 10− 6
mol/ m3 teractions between peptide and vincristine, respectively. Enzymes
CV Initial concentration drug 2.2 mol/ m3 degrade the matrix and change bound species into free ones (see Fig. 6).
Csolvent Concentration of solvent (water) 5.5 × 10− 4 mol/ m3 Each species is represented by a mass balance equation. In this
DBV Diffusion coefficient of matrix-bound vincristine, 24.95 × 10− 11
m2/s equation, there are some terms to describe association, dissociation, and
biomaterial matrix (Saltzman et al., 1994)
diffusion (only for diffusible species). To illustrate the degradation by
DBP Diffusion coefficient matrix-bound peptide, 0 m2/s
Vmax [S]
biomaterial matrix (Saltzman et al., 1994) enzymes, the Michaelis-Menten term (Bhagavan and Ha, 2011) KM +[S],
DBPV Diffusion coefficient of matrix-bound vincristine- 0 m2/s
was used in Eqs. (10)–(13). In this term, S represents concentration of
peptide species, biomaterial matrix (Saltzman
et al., 1994)
the substrate, Vmax is the maximum rate, and KM is the Michae­
DFP Diffusion coefficient of vincristine, biomaterial 24.95 × 10− 11
m2/s lis–Menten constant. By adding this term to mass balance equations, the
matrix (Saltzman et al., 1994) effect of degradation on species concentration can be determined.
DFPV Diffusion coefficient of vincristine-peptide, 2.34 × 10− 11
m2/s Mass balances are expressed in the form of partial differential
biomaterial matrix (Saltzman et al., 1994)
equations for every species (Maxwell et al., 2005; Dolatabadi et al.,
Denzyme Diffusion coefficient enzyme, biomaterial matrix 3.3 × 10− 11
m2/s
(Saltzman et al., 1994) 2021b):
DSD Diffusion coefficient of vincristine, surroundings 27.72 × 10− 11
m2/s
∂ci ( )
(Saltzman et al., 1994) + ∇. − Di,k ∇ci = Ri,k . (8)
DSP Diffusion coefficient of peptide, surroundings ( 2.6 × 10− 10
m2/s ∂t
Saltzman et al., 1994)
DSPV Diffusion coefficient of vincristine-peptide 2.36 × 10− 11
m2/s here Di,k , ci and Ri,k represent diffusion coefficient, concentration and
species, surroundings (Saltzman et al., 1994) release rate of speciei, in medium k (biomaterial matrix or surrounding),
DSenzyme Diffusion coefficient of enzyme, surroundings ( 3.66 × 10− 11
m2/s respectively.
Saltzman et al., 1994)
According to the reaction network as depicted in Fig. 6, the following
equations can be employed for each of the species present in the system
Hydrogel swelling is predominantly caused by the osmotic pressure. (biomaterial matrix) (Maxwell et al., 2005).
Following are the equations relating the swelling ratio and osmotic RV,k = − kf cV (cBP + cP ) + kr (cBPV + cFPV ) (9)
pressure, as functions of ions concentration (Bayat et al., 2020a):
∑ RFP,k = − kf cV cP + kr cBPV + RMMBP (10)
Πosm ≃ Πion = RT (ck − c0k ),
k
RFPV,k = kf cV cP − kr cFPV + RMMBPV , (11)
Js = ξΠosm , (5)
RBP,k = − kf cV cBP + kr cBPV − RMMBP , (12)

5
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

Fig. 7. Calibrating isochoric material parameter of Eq. (4) with experimental data. Dotes express the experimental data and solid lines show the calibrated numerical
relations. Experimental data for e1 and e2 were obtained from Eqs. (6) and (7).

RBPV,k = kf cV cBP − kr cBPV − RMMBPV (13) 3.3. Coupling deformation and diffusion

where RMMBP and RMMBPV represent degradation of BP and BPV matrices
according to Michael-Menten kinetics, and describe dissolving of BP and
BPV species and generation of P and PV species instead, respectively.
Vmax cBP
RMMBP = , (14)
KM + cBP
To determine the diffusion coefficient, the following equation was
used to relate the diffusion behavior to mechanical properties (Wilmers
and Bargmann, 2015). This equation is a function of volume ratio and
concentration. In this study the temperature was considered to be fixed.
D(J, c) = D0 [1 − ϕS ]2 [1 − 2χ ϕS ], (23)

cV
Vmax cBPV
RMMBPV = . (15) where ϕS = vvS = JV
ρs
= Jcρ is the volume fraction of solvent, c is the current
s
KM + cBPV
solvent concentration. Here D0 is the initial diffusion coefficient. Thus,
To set initial boundary conditions, the initial degradation is assumed the volume fraction can be obtained based on concentration of different
zero, thus, one may write: speices over time and volume ratio (J).
kf cV cBP = kr cBPV , (16) To investigate the effect of degradation on the free energy (Guo and
Sluys, 2006); the Mullins effect was added to the model which di­
cTOT = ceq eq
(17) minishes the deviatoric energy in Eq. (5) by multiplying it in an ever-
V + cBPV ,
decreasing function. Hence, the isochoric strain energy equation is
V

cTOT = ceq eq
(18) recast as:
P BP + cBPV .

In the surrounding medium, only association and dissociation exist.
Therefore, the boundary conditions are:
RFV,k = RFP,k = − kf cV cP + kr cPV ,
RFPV,k = kf cV cP − kr cPV .
All variables/constants are given in Table 1. COMSOL-Multiphysics
was employed to model and solve these coupled nonlinear equations.
Transport of diluted species physics were added for the biomaterial
matrix and surrounding medium and coefficients of mass balance
equations and other initial values were defined for each of the species.
Molar concentration of species were calculated by dividing the uti­
lized mass concentration to their molecular weight (Petrucci et al.,
2002). To determine initial diffusion coefficients, the following equa­
tions were used for each species (Saltzman et al., 1994):
Dwater = AMw− 1/3 × 10− 7
D0gel = 0.9 × Dwater
where Dwater denotes the diffusion coefficient of species in water and
D0gel denotes the initial diffusion coefficient in gel. A is a constant value
(A = 260 cm2/(s × Dalton)), and Mw is the molecular weight of species.
Using the correlation for diffusion within porous hydrogels reported in
Saltzman et al. (1994); Dwater was multiplied by 0.9 to obtain the initial
diffusion coefficient in the gel.
ψ iso = (1 − d)ψ iso (24)
where d is the damage variable which is in the range of (WHO, 2018). In
(19) order to define d, Miehe’s damage evolution function is utilized (Miehe,
1995):
(20) ( )
ψ iso,max − ψ iso
d = d∞ [1 − exp − ] (25)
ψ m,sat
in which d∞ and ψ m,sat are model parameters representing the maximum
damage and damage saturation energy, respectively.
4. Results and discussion
The purpose of this research was to propose a comprehensive model,
based on experimental results, to predict the drug delivery behavior of
silk fibroin hydrogel loaded with vincristine as a therapeutic material. In
(21)
this regard, a mechanical model was developed based on stretching and
mixing free energy. Then, the chemical behavior of hydrogel and drug
(22)
species was investigated by considering the degradation behavior and
interactions between the matrix and drug. Finally, to couple the diffu­
sion and mechanical behavior, a diffusion coefficient equation which is a
function of volume ratio J, was added. Employing all the above-
mentioned equations and performing the calibration based on the ex­
periments, the drug release behavior of hydrogel was studied. In the next
step, simulation results were compared to the experimental ones to

6
Z. Gharehnazifam et al.
Fig. 8. (a) Drug release profiles through the surrounding domain at various selected times of updating the content of the well with 4.8 mm radius. (b) Drug release
profiles through the surrounding domain at various selected times in a well with 4.8 mm radius, without updating the content.
verify its validity. As part of the experiments, only the deswelling of
hydrogel based on its weight was examined, along with the drug release
rate based on the UV test. The cumulative concentration of drugs was
validated against the experimental data. Continuity of the species con­
centration is an assumption. However, this is not far from reality, since
the distribution coefficient (or partitioning coefficient) for each species
depends on the medium properties which is not that different in this
study and can be simplified to unity. In the following, the achieved re­
sults are depicted and discussed in detail.
The calibration procedure and experimental data in Appendix A were
used to obtain the material parameter needed for hyperelastic free en­
ergy calculations. Substituting this parameter in the free energy Eq. (4),
the model is able to well predict the experimental data, as depicted in
Fig. 7. Material parameter c1 was computed as 1.5982 × 105 Pa.c2 was
found by calibrating with the swelling diagram. The standard deviation
for the results of e1 is 0.01 J/ m3 and this deviation for the results of e2 is
0.02 J/ m3.
These mathematical manipulations were coded in the Python lan­
guage. Then, material parameters and the free energy equation were
used as input in COMSOL-Multiphysics to develop the proposed model
in simulations.
The geometry of hydrogel was considered as a cylinder with radius of
1.3 mm and height of 10 mm based on the approximate shape after
injection which was measured by a gauge caliper. The release profile
was investigated in times right before depleting the wells, as depicted in
Fig. 8a. It depicts the concentration of drug through a horizontal cut line
7
International Journal of Pharmaceutics 609 (2021) 121184
which is tangent to the upper surface of the hydrogel cylinder, at the
selected times up to 40 days. Around the matrix, the drug concentration
is maximum and by getting away from the matrix, this concentration
decreases. It shows a burst release in the first hour of the drug release,
while it diminishes over time. In longer periods between PBS solution
removals (1 week), the drug concentration will be uniformed through
the medium. Fig. 8b illustrates the release profile when the content of
wells is not replaced after specified periods. It shows that after 40 days,
about 70% of the drug is released, while the release amount after day 5 is
almost negligible. A decrease in release rates is due to a rise in the os­
motic pressure within the medium. Thus, the content of the medium
should be updated periodically. This figure also shows that all the
release profile curves intersect at a certain distance from the hydrogel.
Thus, the concentration there remains almost constant over time. Hence,
it is possible to design it so that a specified area has a specified con­
centration range.
In Fig. 9, the release behavior at the selected times is depicted in 3D
modeling domain. As mentioned before, these selected times are the
periods defined to remove the content of the medium because of the
osmotic pressure. In the first hour, the drug release rate is maximum,
however it is not yet distributed completely inside the medium. The drug
release rate decreases over time and is distributed uniformly inside the
medium. Fig. 9 shows that through the bigger side of the matrix, the
drug concentration is higher than on other sides. Besides, in the corners,
the drug concentration is minimal. Based on this result, the optimum
time between different injections and optimum coordinate for multi-
Z. Gharehnazifam et al.
Fig. 9. Drug concentration (mol/m3) in the surrounding medium at selected times. a: Time = 0 h, b: Time = 1 h, c: Time = 1 d, d: Time = 5 d, e: Time = 12 d, f: Time
= 19 d, g: Time = 26 d, h: Time = 33 d.
injections can be predicted for in vivo applications.
Experimental results of the drug release from silk fibroin hydrogel in
PBS (no enzyme) show a continuous release pattern. After 40 days, only
10% of the drug is released from the hydrogel. It is due to the absence of
any enzyme in the medium along with the degradation effect. As one
may observe from Fig. 10, in the enzyme solution, a burst release is
visible in the early hours of release. After about 5 days, the release rate is
negligible and most of the drug has been released. The standard devia­
tion for the results of cumulative release in PBS solution is 1.36% and
this deviation for the enzyme-based solution is 2.7%. Comparing the
modeled release to the experimental ones shows a good agreement,
where the difference stems from geometry of the hydrogel, affinity be­
tween the drug and gel, as well as other surrounding conditions.
The experimental studies on swelling behavior reveal that the
deswelling ratio of silk fibroin hydrogel in PBS solution is negligible (see
Fig. 12). A similar observation was reported in experiments of Xu et al.
(2019) confirming this behavior for nearly pure silk fibroin hydrogel.
8
International Journal of Pharmaceutics 609 (2021) 121184
These results inspired some simulation studies to compare the results
with. In order to assess the deswelling behavior in simulations, the
concentration of different species is tracked over time in Fig. 11. A
swelling response is affected by osmotic pressure, which depends on
species concentration.
Enzyme type II collagenase causes degradation of matrix and matrix
bound species; thus, as illustrated in Fig. 11a, the concentration of
different species in the medium varies over time, where the matrix de­
grades after about 40 days. Matrix bound species concentration (BP,
BVP) decreases and free peptide species concentration (FP, FVP) in­
creases with time. This model was checked for a state that enzyme
concentration is zero (enzyme free solution) and the result is depicted in
Fig. 11b, no degradation is observed after 100 days.
To better analyze and compare the simulation and experimental re­
sults of swelling response, Fig. 12 is presented. When type II collagenase
enzyme was added to the medium, the degradation phenomenon trig­
gered a higher release rate. Simulated swelling profiles were derived by
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

Fig. 10. Cumulative drug release from silk fibroin hydrogel in experimental and simulated models. The continuous lines represent simulated results and the dots
represent the experimental results in pure PBS and enzyme solution. SD shows the standard deviation for simulation results.

Fig. 11. (a) Concentration of different species (mol/m3) in an enzyme solution as a function of time (day). (b) Concentration of different species (mol/m3) in PBS
solution as a function of time (day).

9
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

Fig. 12. Swelling ratio of silk fibroin hydrogel in PBS and type II collagenase solution (pH = 7 and T = 37 ◦ C).

Fig. 13. (a) Rheometery results for storage and loss module (G’ & G”) in various shear strains, (b) Converted rheometery data into energies using Eqs. (6) and (7).

dividing the volume ratio (J) by the initial swelling ratio (Js). The initial
swelling has been calculated based on the amount of dry silk and water.
As demonstrated in Fig. 12, while silk fibroin takes approximately 40
days to completely deswell in enzyme solution, this process is negligible
in PBS solution since the species concentration is constant over time. The
standard deviation for deswelling ratio in PBS is 0.03 and this deviation
for the deswelling in enzyme-based solution is 0.1. There is an accept­
able agreement between the simulated and experimental results, and the
deviation can be related to the surrounding conditions or precision of
the weighting process.

10
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

5. Summary and conclusion
In this study, the release behavior of silk fibroin hydrogel was
investigated through experiments and developing a computational
model. Material parameters of silk fibroin were determined by experi­
mental rheology results. In order to have a comprehensive study of the
drug release behavior, a coupled model of affinity-based diffusion,
degradation and mechanical behavior was considered. COMSOl-
Multiphysics software was used for simulation studies.
The free energy equation was calibrated with experimental data
using a reverse method and linear regression, then applied in numerical
simulations carried out in COMSOL-Multiphysics to get a better insight
into the behavior of hydrogels. Chemical reaction simulations revealed
that the hydrogel deswelled in type II collagenase enzyme solution after
40 days, but not in the pure PBS solution. Similar results were observed
in experiments, as well. The drug was released dramatically within the
first hour. The release rate declined over time. By replacing the content
of the medium, the drug was released completely after 40 days, but
when no content replacement was made, the maximum release rate did
not exceed 70%. In this type of release, the concentration remained
constant over time at some points; thus, the system can be designed to
keep the concentration fixed for a particular target region. The results of
3D simulation revealed that the drug concentration is higher on the
larger side of the matrix and minimum at the corners. These results can
be used to determine the optimal timing for the injection and the co­
ordinates for multi-injections in vivo.
Based on experiments and simulations, cumulative release behaviors
showed that in pure PBS solution, drug release did not exceed 10% after
40 days, but in enzyme-containing solution most of the drug was
released within 5 days. In addition, the simulation results were in good
agreement with the experimental results. Therefore, this hybrid model
can be implemented to predict drug release characteristics of hydrogels
and to adjust injection times as well as coordinates.
CRediT authorship contribution statement
Ziba Gharehnazifam: Methodology, Writing – original draft.
Roshanak Dolatabadi: Methodology, Writing – original draft. Majid
Baniassadi: Conceptualization, Methodology. Hamid Shahsavari: .
Abdol-Mohammad Kajbafzadeh: . Karen Abrinia: Conceptualization,
Methodology. Mostafa Baghani: .
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This work is supported by the Pediatric Urology and Regenerative
Medicine Research Center of Tehran, University of Medical Sciences,
with the grant number of 38291-84-02-97, and ethical committee
permission number of IR.TUMS.VCR.REC.1397.530.

Appendix A. Further comments on reverse method

A linear regression is used to determine the material parameters of the free energy function, as discussed in Section 3.1. In the following, the
procedure of finding these parameters is described (Abali et al., 2019). The rate of energy can be written as follows:

ψ̇ = c1 I˙ 1 (A1)
Strain in an oscillatory rheometer with the frequency of ν, is described as:
ε = ε0 sin(2πνt) (A2)

where ε0 is the strain amplitude. Thus, shear deformation is considered as:

⎛ ⎞
1 ε 0
Fij = 0 1 0 ⎠
⎝ (A3)
0 0 1

Thus, right Cauchy–Green strain tensor C = FTF, becomes:

⎛ ⎞⎛ ⎞ ⎛ ⎞
1 0 0 1 ε 0 1 ε 0
Cij = ⎝ ε 1 0 ⎠⎝ 0 1 0 ⎠ = ⎝ ε 1 + ε2 0 ⎠ (A4)
0 0 1 0 0 1 0 0 1

in which e1 and e2 can be defined by experimental values of G′ and G′′ . For e1 and e2, the following equations can be obtained for a complete period and
a quarter of a period, respectively.
e1 = A11 c1 (A5)

∫T
A11 = I˙ 1 dt. (A6)
0

e2 = A21 c1 (A7)
T
∫4
1
A21 = I˙ 1 dt − A11 . (A8)
0 4
Then, by solving the following equations, the material parameters will be calculated.

11
Z. Gharehnazifam et al. International Journal of Pharmaceutics 609 (2021) 121184

⎛ ⎞
⎛ ⎞ ⎜ ⎟
⎜ ⎟
⎜ ⎟ ⎜ ⎟
⎜ ⎟ ⎜ 2π G˝| 1 (ε1 )2 ⎟
⎜ ⎟ ⎜ ε0 0 ⎟
⎜ A11 |ε1 ⎟ ⎜ ⎟
⎜ 0
⎟ ⎜ G’| (ε1 )2 ⎟
⎜ ⎟ ⎜ ε1 0 ⎟
⎜ A21 |ε1 ⎟ ⎜ 0 ⎟
⎜ 0 ⎟ ⎜ ⎟
⎜ ⎟ ⎜ 2π G˝| 2 (ε2 )2 ⎟
⎜ A11 |ε2 ⎟ ⎜ ε0 0 ⎟
⎜ ⎟ ⎜ ⎟
⎜
0
⎟ ⎜ 2
⎟
⎜ ⎟
⎜
⎜
A21 |ε2 ⎟(c1 ) = ⎜ G’|ε2 (ε20 )
⎟ ⎟ (A9)
⎜
0
⎟ ⎜ 0
⎟
⎜ ⎟ ⎜ ⎟
⎜ ⋮ ⎟ ⎜ ⋮ ⎟
⎜ ⎟ ⎜ ⎟
⎜ ⎟ ⎜ ⎟
⎜ A11 |εn ⎟ ⎜ 2π G˝| n (εn )2 ⎟
⎜ 0
⎟ ⎜ ε0 0 ⎟
⎜ ⎟ ⎜ ⎟
⎜ A21 |εn ⎟ ⎜ ⎟
⎜ ⎟ ⎜ ⎟
⎝
0
⎠ ⎜ G’|εn (εn0 )2 ⎟
⎜ 0 ⎟
⎜ ⎟
⎝ ⎠

whereε10 , ε20 , … , εn0 are different strains for n measurements at a specific frequency.
The above equations can be summarized as:
Ac = b, (A10)

where c is unknown and describes material parameters, b denotes experimental results and A is the coefficient matrix. This equation set is linear and
the answer is not unique; hence, it can be solved employing the least square error method. This equation set can be solved as the following:

c = (AT A)− 1 AT b (A11)

As discussed earlier, to determine the material parameters of the hyperelastic energy function, rheology results were utilized. Fig. 13a demon­
strates these results for silk fibroin hydrogel at the frequency of 1 Hz and various shear strains (up to 1%). Consequently, in light of Eqs. (6) and (7),
these diagrams should be converted to the stored and loss energy (respectively e1 and e2). Fig. 13.b shows the converted diagrams for e1 and e2. These
results are required in constructing b in Eqs. (A10) and (A11).
Matrix A (in Eqs. (A10) and (A11)) was computed by Simpson’s rule to integrate Eqs. (A6) and (A8). Then, material parameter c, was found from
solving Eqs. (A10) and (A11).

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