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Division of Pharmaceutics, Department of Pharmaceutical Technology and ∥Bioequivalence Study Centre, Department of
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Table 1. Different Batches of Carboxymethyl Pullulan, Their Conditions of Preparation, Degree of Substitution Values, and
Elemental Analysisa
process variables elemental analysis
batch code volume of 10 M NaOH (mL) MCA (g) temp (°C) DS C (%) H (%) O (%)
NP 41.06 7.72 51.22
CMP-1 5 5 65 0.296 ± 0.056 36.79 6.75 56.46
CMP-2 10 5 65 0.539 ± 0.032 30.91 5.29 63.80
CMP-3 15 5 65 0.395 ± 0.029 35.28 5.82 58.90
CMP-4 20 5 65 0.328 ± 0.028 35.22 6.46 58.32
CMP-5 5 2.5 65 0.157 ± 0.025 38.22 7.69 54.09
CMP-6 10 2.5 65 0.345 ± 0.028 35.64 6.35 58.01
CMP-7 10 5 95 0.433 ± 0.030 34.97 6.62 58.41
CMP-8 10 5 35 0.282 ± 0.027 36.91 7.29 55.80
CMP-9 2.5 2.5 65 0.116 ± 0.024 39.11 7.44 53.45
CMP-10 7.5 2.5 65 0.250 ± 0.027 36.05 7.29 56.66
CMP-11 12.5 2.5 65 0.314 ± 0.000 36.07 6.56 57.37
CMP-12 15 2.5 65 0.298 ± 0.027 36.29 7.01 56.70
CMP-13 10 7.5 65 0.505 ± 0.031 33.29 7.02 60.69
a
NaOH: sodium hydroxide; MCA: monochloroacetic acid; temp: temperature; DS: degree of substitution.
tration for a longer duration of time.3 In the last few decades, suppression by opioids in IPF patients, while pirfenidone
application of natural polymer in controlled drug-delivery (PFD) has been proved to be effective with beneficial effects
system has gained a significant importance in medical and on a subgroup population suffering with IPF.22
biomedical applications.6,7 The various different polymers Our aim is to develop a therapeutically effective controlled
harnessed include xanthan gum7 chitosan,8 gellan gum,9 locust drug-delivery system for the symptomatic management of IPF,
bean gum,10 guar gum,11 etc. In most reports, it was observed which will be able to release the required amount of drug as
that the in vitro release of drug(s) from the polymeric matrix loading dose and rest of the drug in equivalent rate as
sustained more than 24 h.12−15 But the residence time of any maintenance dose. As PFD is used in management of dyspnea
solid oral formulation in gastrointestinal tract is as maximum as and cough in IPF patients, the time taken to achieve peak
8−10 h depending on the fed and fasted conditions unless the plasma concentration (tmax) should be as low as possible to get
formulation is modified for specific targets. Therefore, a large immediate relief from the dyspnea and cough. In a study
amount of drugs still remains in the formulation matrix after conducted by the sponsors for approval filing to U.S. Food and
8−10 h, which could not be available for absorption, meaning Drug Administration, it was found that 45.80 ± 8.24% of the
underutilization of delivered dose.4 Hence, there is a need to 14
C PFD dose was absorbed from the stomach within 30
control the drug release from a rate-controlling polymer matrix min.23 Hence, the swelling behavior of polymeric matrix must
within certain hours (8−10 h) to achieve maximum absorption be pH-dependent so that the release of drug from the matrix in
of drug. The amount of rate-controlling polymer used in stomach (acidic pH) would be sufficient to meet the criteria of
formulation is not only the factor that controls the release rate, loading dose. Polymeric matrix must also be able to release the
but other factors such as swelling properties, network cross- rest of the drug in intestine as to suffice the maintenance dose
linking sites, and density plays a crucial role to achieve within the transit time of approximately 6 h. The swelling
preprogrammable release rate within a certain time peri- characteristic of IPN microspheres using carboxymethylated
ods.16−19 Accordingly, there is a need to tailor the native polymer and poly(vinyl alcohol) (PVA) was studied by
polymer to incorporate the required properties. different scientists, which confirmed pH-dependent swelling
Interpenetrating polymeric network (IPN) is one such behavior, indicating a higher swelling index in alkaline
innovative form of drug-delivery system, which comprises two medium.19,24,25 So, to develop IPN microsphere of PFD with
or more networks, partially interlaced on a molecular scale, similar characteristics, the native biopolymer pullulan was
which cannot be separated until the chemical bonds are modified to carboxymethyl pullulan (CMP), to render it pH-
broken.20 Hence, there are many variables available in the IPN sensitive before preparing IPN microsphere. The pH sensitivity
system, which ultimately gives us the opportunity to achieve is a prerequisite, as swelling followed by drug release will be
intended specific requirements. Apart from modification of the affected by pH of the media.
polymer used as the backbone in IPN system, other variables Microsphere was prepared using glutaraldehyde (GA)-
such as the amount of cross-linker and, more specifically, drug assisted water-in-oil (w/o) emulsion cross-linking technique.
polymer ratio will also be available to control the release rate Spectral characterization was performed to confirm the cross-
within the transit time period.21 linking and formation of IPN microsphere. Field emission
Idiopathic pulmonary fibrosis (IPF) is a chronic, cata- scanning electron microscopy (FESEM) was performed to
strophic, and progressive lung disease with very-difficult-to- study the shape and surface morphology of microspheres. In
treat situation, leading to respiratory failure and death within vitro enzymatic degradation was performed to assure the
3−5 years of diagnosis. The initial symptoms of IPF are degradability of microspheres. Biocompatibility study and cell
dyspnea, cough, fatigue, depression, anxiety, relationship viability study were conducted on human hepatocellular
problem, and financial difficulties, of which dyspnea and carcinoma (HepG2) cell lines using 3-(4,5-dimethylthiazol-2-
cough are the serious issues for which management is yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell
immediately required. There is no evidence of cough imaging techniques. In vitro release profile and release kinetics
11994 DOI: 10.1021/acsomega.8b00803
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Table 2. Weight Average Molecular Weight, Viscosity, Contact Angle, and Surface Energy Measurement of Native Pullulan and
Carboxymethyl Pullulana
batch code Mw (g/mol) PDI viscosity (cP) contact angle (deg) surface energy (mN/m)
NP 8.51 × 105 1.922 98.50 73.04 ± 0.75 39.64 ± 0.69
CMP-1 8.10 × 106 1.288 64.80 43.41 ± 0.12 57.47 ± 0.00
CMP-2 4.47 × 106 1.163 60.20 33.7 ± 6.6 62.6 ± 3.3
CMP-3 6.94 × 106 1.144 62.10 57.26 ± 0.12 49.45 ± 0.04
CMP-4 1.30 × 107 1.006 61.40 58.5 ± 1.3 48.73 ± 0.80
CMP-5 1.11 × 106 1.277 69.40 63.99 ± 0.21 45.40 ± 0.13
CMP-6 9.14 × 105 1.126 68.10 55.5 ± 3.3 50.4 ± 1.7
CMP-7 1.72 × 106 1.719 60.50 56.3 ± 1.2 49.5 ± 1.5
CMP-8 4.30 × 106 1.002 62.90 63.6 ± 1.8 45.98 ± 0.59
CMP-9 1.17 × 107 1.318 65.70 71.2 ± 1.8 40.9 ± 1.4
CMP-10 1.39 × 106 1.219 66.40 62.7 ± 2.1 46.2 ± 1.3
CMP-11 1.47 × 105 1.718 62.50 61.15 ± 0.37 47.05 ± 0.62
CMP-12 1.26 × 106 1.316 63.10 67.4 ± 1.7 45.79 ± 0.32
CMP-13 5.10 × 107 1.193 61.10 45.1 ± 2.7 57.5 ± 1.4
a
Mw: weight-average molecular weight; PDI: polydispersity index; cP: centipoise; deg: degree; mN/m: millinewtons per meter.
of all formulations (F1−F9) were explored. Finally, optimized effect of temperature on DS value was also studied, which
formulation and marketed product (Pirfenex) were subjected showed the highest DS value (0.539) at 65 °C and reduced
to an in vivo pharmacokinetic (PK) study in rabbits and values at 35 °C (0.282) and 95 °C (0.433) (Figure S2c,
different PK parameters were compared, showing that the Supporting Information). The reduced DS value at reduced
microsphere was able to control release for 12 h, with Cmax temperature was observed due to less swelling of polymer and
achieved in 0.83 h, satisfying our goal. In this way, the lack of activation energy to initiate and drive the reaction,
developed formulation could be a suitable candidate for while increased temperature caused chain degradation and
therapeutically effective controlled release formulation of PFD. intramolecular elimination of water, leading to a reduced
number of hydroxyl groups available for substitution.26 Thus,
2. RESULTS AND DISCUSSION considering the above observation, reaction condition was
2.1. Synthesis of Carboxymethyl Pullulan and Effect optimized based on the highest DS value.
of Various Process Variables on Degree of Substitution 2.2. Formulation of PVA-CMP IPN Microspheres. In
(DS). Pullulan is composed of a linear chain of maltotriose the present work, IPN microspheres were prepared using the
units linked by α-(1 → 6) glycosidic bond and possess various (w/o) emulsion cross-linking technique. The synthetic
hydroxyl groups in its structure, which are desirable for biopolymer PVA was blended with CMP and interlaced on
substitution of carboxymethyl groups. The reaction pathway the backbone of CMP with the help of GA, a bifunctional
for the synthesis of CMP is illustrated in Figure S1, Supporting chemical cross-linker, which formed acetal bond between the
Information. hydroxyl group of PVA and CMP. This resulted in the
All of the process variables were taken into consideration to formation of interpenetrating polymer network, a three-
maximize the DS as per Table 1. dimensional (3D) structure of hydrogels, which can be
2.1.1. Effect of Process Variables on DS. Degree of distinguished from the regular polymeric blends.28 The
substitution is the initial and main characterization parameter prepared microspheres were insoluble in water and showed
for a successful carboxymethylation reaction. DS value was controlled hydration property with drug entrapped in the
found to be highly dependent on the concentration of sodium polymeric network. The reaction pathway for the formation of
hydroxide, MCA, and reaction temperature, as can be observed IPN microsphere is presented in Figure S3, Supporting
from Table 1 and Figure S2, Supporting Information. With Information.
increase in the concentration of sodium hydroxide up to 10 2.3. Physicochemical Characterization. 2.3.1. Elemental
mL, the DS value increased (DS = 0.539) and then started to Analysis. The presence of different elements (C, H, and O) in
decrease with further increase of NaOH concentration (15 and native pullulan and CMP was determined, and the results are
20 mL, DS = 0.395 and 0.328, respectively). The initial presented in Table 1. The results showed that there was a
increase in DS value was due to the activation of polymer for significant increment in oxygen content in all of the batches of
the formation of alkoxide and facilitated the swelling of CMP compared to native pullulan and highest for CMP-2
pullulan for enhanced diffusion and penetration of etherifying (63.80%). This observation was due to the attachment of
agent (MCA). The DS value was reduced on further increase −CH2COONa group at the replaceable hydroxyl group of
of NaOH concentration due to the competition between the pullulan. The carbon and hydrogen contents were found to be
main reaction and the side reaction of NaOH and MCA, with reduced for CMP (C = 30.91%, H = 5.29%, for CMP-2)
the side reaction becoming predominant with increased compared to native pullulan (C = 41.06%, H = 7.72%) due to
production of glycolate.26 The effect of MCA concentration polymer chain distortion because of high concentration of
was also studied, and at optimized concentration of NaOH, the NaOH, vigorous stirring during the reaction, and participation
DS value initially increased with increasing MCA concen- of hydrogen in the formation of water molecules.
tration and then reduced on further increase in MCA 2.3.2. Viscosity Study. The viscosity of native pullulan was
concentration (Figure S2b, Supporting Information), due to found to be 98.50 cP, which is higher compared to all CMP
increased formation of glycolate with increased MCA.27 The batches (69.40−60.20 cP), as evident in Table 2. The reduced
11995 DOI: 10.1021/acsomega.8b00803
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Figure 3. 13C NMR spectra of (a) carboxymethyl pullulan and (b) placebo PVA-CMP IPN microsphere.
single-step major degradation of 67.32% in the temperature observation was due to heat transfer lag in the heating rate.38
range of 507−630 K, supported by DTG curve showing the Earlier, we had reported the thermal analysis of pullulan
sharp peak of degradation at 552 K at a heating rate of 5 K/ performed with similar methodology, which showed a single-
min. The degradation peak of CMP at different heating rates 5, step sharp degradation peak at 590 K. The observed difference
10, 15, and 20 K was found to be slightly shifted toward higher suggested structural changes during carboxymethylation;
temperatures 552, 564, 567, and 578 K, respectively, as shown furthermore, the presence of sodium ion of sodium
in the DTG curve of Figure S7, Supporting Information. The carboxymethyl group in the CMP also alters the thermal
11998 DOI: 10.1021/acsomega.8b00803
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Figure 4. FESEM images of (a) pullulan, (b) carboxymethyl pullulan, (c) group of microspheres, (d) single microsphere, and (e) cross-linked
network in the polymer matrix.
decomposition pattern,39 which confirmed successful mod- breakage of polymeric chain into smaller units with elimination
ification of pullulan. of the hydroxyl group. The third step included breakage of
The TGA and DTG curves of PVA were reported in our carbon chains (C−C), as evident with the sharp degradation
previous study,21 which indicated a sharp degradation peak at peak at 706 K (682−718 K), and the fourth degradation zone
600 K due to rapid chain fastening and elimination of H2O, 718−789 K indicated carbonization and formation of tars of
converting the polyenes to aliphatic group by Diels−Alder carbon units.
intramolecular cyclization or radical reactions. Upon progress The thermal analysis of pure drug PFD showed a single-step
of reaction, the C−C cleavage and its carbonization occurred degradation at 510 K at a heating rate of 5 K/min degradation
at 720 and 750 K, respectively.40 range (429.28−515 K), which was shifted to higher temper-
The TGA and differential thermal analysis (DTA) curves of ature with increase in heating rate (Figure S11, Supporting
IPN microsphere showed different degradation patterns from Information).
its individual components. In the case of microsphere, four Thermogram of optimized formulation (F5) showed a
degradation zones were observed in the range of 300−850 K. similar pattern to placebo microsphere (Figure S12, Support-
The first degradation was observed as a small hump in the ing Information). The degradation zone of drug was clearly
range of 363−435 K with a nominal weight loss of 4.83%, visible in the range of 427−544 K, but the degradation pattern
which could be due to loss of moisture entrapped in the and zones remain the same, indicating the stability of the IPN
polymeric network. The second zone of degradation was from matrix in the presence of drug.
526 to 678 K, with a weight loss of 55.47%, indicating the This thermal observation supported the formation of IPN
major degradation zone. The DTG curve in this range showed microsphere, and their TGA and DTG curves at different
a broad peak at 647 K for lower heating rates (5 and 10 K/ temperatures are shown in Figures S8−S10, Supporting
min), which became comparatively sharper for heating rates Information.
(15 and 20 K/min) due to increased heating rate. This 2.4.2. Activation Energy. Activation energy (Ea) was
indicated the formation of IPN microsphere as the degradation calculated using the Ozawa−Flynn−Wall (OFW) and Kis-
zone of CMP (507−630 K) and PVA (495−650 K) broadened singer−Akahira−Sunose (KAS) models. These models yielded
and shifted in the range of 526−678 K, which included rapid a slope value (Figure S13, Supporting Information) from the
11999 DOI: 10.1021/acsomega.8b00803
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linear relationship between ln(β) vs 1/T (eq 2) and the slope degradation of polymeric chain in this region, and then
(Figure S14, Supporting Information) of graph ln(β/T2) vs 1/ significantly decreased, as evident from Figure 5.
T when multiplied with gas constant (R = 8.314 J/(mol K)). IPNs showed an increasing trend in the activation energy
Slope activation energy as a function of degree of conversion with increasing conversion factor (α). The activation energy
(α) for all of the samples is graphically shown in Figure 5. was minimum at α = 0.1 and increased up to α = 0.7, which
showed that high energy was required with increasing step for
polymeric chain cleavage due to the formation of cross-linked
network structure (formation of IPN), and then decreased at α
= 0.8, indicating decomposition of almost polymeric chain and
network. At this stage, different pyrolysis byproducts
(dehydrated products of carbon structure) like alkane, alkene,
dienes, and aromatic cyclization of carbonaceous char residues
were formed and required high activation energy for its
conversion to carbon tars as evident at α = 0.9, in Figure 5, and
at this stage, weight loss was about 99% and no residue was
left.41 This kind of observation was consistent with all of the
three IPN microspheres and activation energy increased with
increasing concentration of cross-linker at a fixed conversion
factor.
The activation energy of the optimized formulation was
found to be slightly reduced (Figure 5) in the entire range of α
= 0.1−0.9. The observation may be due to the effect of drug,
so the Ea value of drug was also calculated, which on an average
was found to be 92.49 kJ/mol, much lower than placebo IPNs.
The increasing trend of Ea value with increasing conversion
factor suggests the formation of stable PFD-entrapped IPN
microsphere.
The data fitted in both the models showed almost parallel
regression lines (R2 > 0.99) in the conversion range (0.1 < α <
0.9), indicating the applicability of the OFW and KAS models
for the calculation of activation energy.41 Upon analysis of the
Ea values of both models, the value for the KAS model is lower
than that of the OFW model, which can be due to the different
approximations of the temperature integral. However, both the
models show similar trend without any significant difference on
Ea with conversion factor under consideration (0.1−0.9),
indicating the chosen isoconversional models to be reasonably
true.
2.5. In Vitro Enzymatic Degradation Study. In vitro
action of pullulanase (E.C. No. 3.2.1.41) was studied on CMP
and PVA-CMP IPN microsphere (placebo) to assure the
biodegradation of synthesized novel products. Pullulanase is a
debranching enzyme, which specifically hydrolyzes α-(1 → 6)
Figure 5. Activation energy as a function of the conversion factor (α) glycosidic bond.42 The experimentally obtained data were
using (a) OFW model and (b) KAS model. plotted as a digestibility curve of CMP (Figure 6a) and PVA-
CMP IPN microsphere (Figure 6b). The digestibility curve of
CMP showed faster digestion (45.77% after 8 h) compared to
In our previous report,21 activation energy of pullulan was IPN microsphere (41.97% after 48 h). This indicated a very
calculated, which showed initial higher activation energy due to low digestion rate for IPN microsphere due to the formation of
high energy requirement to initiate the glycosidic bond 3D polymer network during cross-linking reaction, compared
cleavage, and once the polymeric chain started to decompose, to CMP, which was present in its two-dimensional structure
the Ea value started to decrease. CMP showed an entirely and the α-(1 → 6) glycosidic bond was easily accessible by the
different pattern of Ea value at different conversion factors due enzyme.
to harsh condition (use of concentrated NaOH and MCA) The log of slope (LOS) plot for the degradation data was
during carboxymethylation process, causing scissions of the made for CMP and IPN microsphere, which showed two
helical coiled structure at the functional groups and the degradation phases for both the test materials (Figure 6c,d).
glycosidic linkage, resulting in restructuring and substitution of The LOS plot yielded two straight lines after it was fitted in the
carboxymethyl group at the hydroxyl group. Initially, the first-order kinetic equation (eq 11). Degradation rate constant
activation energy for CMP was the minimum at 0.1 (54.46 and (k) in min−1 was calculated from the slope value, and C∞ form
47.33 kJ/mol for OFW and KAS models, respectively) and the the intercept of the straight line equation generated from the
energy required for next 10% degradation (α = 0.2) was almost first-order kinetic equation43,44 (Table S1, Supporting
3 times (138.9 and 129.98 kJ/mol for OFW and KAS models, Information). The digestibility rate constant for CMP was
respectively), which was constant up to α = 0.6, showing major higher than IPN microsphere in both the phases due to the
12000 DOI: 10.1021/acsomega.8b00803
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Figure 6. Digestibility curves of (a) CMP and (b) CMP-PVA IPN microsphere, and logarithm of slope (LOS) plots of (c) CMP and (d) CMP-
PVA IPN microsphere.
Figure 7. Viability of the human hepatocellular carcinoma (HepG2) cells detected by MTT assay at 72 h. (a) Dose-dependent cell proliferation
assay of CMP and direct contact of PVA-CMP IPN microsphere. (b) Rhodamine-phalloidin (stains actin)/Hoechst (stains nucleolus) staining of
cells treated at different concentrations: (i) 1000 μg/mL, (ii) 500 μg/mL, (iii) 50 μg/mL of CMP, (iv) control, (v) phase contrast image of cells
adhered to microsphere, (vi) rhodamine-phalloidin/Hoechst staining of adhered cells on microsphere, and (vii, viii) FESEM images of cell adhered
microsphere at 270× and 2000×, respectively. (Data are presented as mean ± SD (n = 3).) The scale bar for the images (i)−(iv) is 400 μm and for
(v)−(vi) is 1000 μm.
linear structure of CMP in which the α-(1 → 6) glycosidic also evident from the values of final concentration (C∞), as
bond was easily accessible compared to sterically hindered reported in Table S1, Supporting Information. Furthermore,
target sites of microsphere due to network formation. This is the LOS graph yielded a linear regression line (R2 > 0.91),
12001 DOI: 10.1021/acsomega.8b00803
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Table 3. Formulation Variables, % Yield, Particle Size d (0.5), Polydispersity Index, % Drug Entrapment Efficiency, and
Equilibrium Water Uptake in pH 1.2 and 6.8a
equilibrium water
uptake
formulation code GA (mL) % drug loading yield (%) particle size [d (0.5)] (μm) PDI DEE (%) (±SD, n = 3) pH 1.2 pH 6.8
F1 3 45 66.76 956.42 0.905 39.62 ± 0.69 156.41 212.09
F2 1 30 62.77 972.26 0.910 25.84 ± 0.50 193.89 273.57
F3 3 30 74.54 939.12 0.947 29.4 ± 1.9 170.15 219.72
F4 5 30 83.81 822.47 0.884 36.43 ± 0.95 158.33 205.38
F5 5 45 72.24 835.16 0.950 46.72 ± 0.49 139.75 188.89
F6 3 15 86.78 926.87 0.880 27.82 ± 0.62 183.72 223.51
F7 1 45 54.90 989.15 0.877 32.9 ± 2.1 180.53 261.47
F8 5 15 92.96 810.12 0.896 31.45 ± 0.73 161.02 209.35
F9 1 15 75.30 958.09 0.909 22.56 ± 0.19 202.38 305.55
a
GA: glutaraldehyde; PDI: polydispersity index; DEE: drug entrapment efficiency.
confirming the first-order digestibility model to be a suitable The second factor (GA concentration) showed increased yield
predictor for enzymatic degradation in our test materials. of final product, with increased GA concentration. The result
2.6. In Vitro Cytotoxicity Test and Biocompatibility was in good agreement as the increased concentration of GA
Assessment. The modified biopolymer will be used for the enhanced breaking of polymeric chain and exposed more
formulation dosage forms for oral route, so it is essential to hydroxyl group available for cross-linking and formation for
confirm the noncytotoxic and biocompatible nature of the acetal bond between PVA and CMP in the emulsion system.
material. The MTT assay for CMP and PVA-CMP IPN The mathematical relationship generated (in coded factors) for
microsphere was performed at four different concentrations percentage yield was as follows
(1000, 500, 100, and 50 μg/mL) and with direct contact to
microsphere and compared to control. After 72 h of y1 = 75.02 + 9.34x1 − 10.19x 2 − 0.080x1x 2 − 2.20x12
incubation, the results showed no significant difference in the + 1.28x 22 (R2 = 0.9950) (1)
cell viability (data presented as mean ± standard deviation
(SD, n = 3)), as shown in Figure 7a. Biocompatibility 2
The predicted R of 0.9502 is in agreement with the adjusted
assessment was performed by cell imaging technique, which R2 of 0.9887 (p ≤ 0.05).
used fluorescent dye rhodamine-phalloidin for actin staining where y1 is the percentage yield of final product, x1 and x2
and Hoechst 33342 for nucleus staining. These images did not are the quantity of GA (mL) and percentage DL, respectively,
show any remarkable change in cellular morphology compared and its contour plot is shown in Figure S16a, Supporting
to control (Figure 7b-i−iv). FESEM image analysis showed Information.
that cells were adhered and spread onto the surface of 2.8.2. Drug Entrapment Efficiency (DEE). The percentage
microsphere (Figure 7b-vii,viii), indicating the noncytotoxic DEE of the prepared microspheres (F1−F9) was calculated
and biocompatible nature of the biopolymer and its micro- and found in the range of 22.56 ± 0.19 to 46.72 ± 0.49%,
sphere. presented in Table 3. The entrapment efficiency of the
2.7. Acute Oral Toxicity Study. The hematological and microsphere was found to be increasing with increasing
serum biochemical analysis reports are presented in Tables S2 concentration of GA at similar % DL (at 15% DL, F9 < F6
and S3, Supporting Information. These results indicated < F8; at 30% DL, F2 < F3 < F4, and at 45% DL, F7 < F1 < F5)
normal levels of hematological and serum biochemical values shown in Table 3. The observation was due to the
parameters, and no significant deviation was observed in test reason that increased concentration of GA increased the cross-
animals compared to control animals. The histopathological linking density, producing highly cross-linked polymer net-
images of the vital organs like heart, liver, kidney, and stomach works, thus preventing the leaching of drug from polymer
did not showed any significant difference (Figure S15, matrices. The increased drug content in the emulsion system
Supporting Information). Further zero mortality of the animals also increased DEE at the similar concentration of GA, due to
were observed, indicating the LD50 value of CMP and PVA- more availability of PFD in the system for cross-linking.45 The
CMP IPN microsphere to be greater than the recommended mathematical relationship generated (in coded factors) for
dose (2000 mg/kg) of body weight. Therefore, CMP and drug entrapment efficiency is as follows
PVA-CMP IPN microsphere were classified under “category 5”
with “zero toxicity” indicating safe for drug-delivery application y2 = 29.95 + 5.56x1 + 6.23x 2 + 1.24x1x 2 + 0.58x12
through oral route. + 3.17x 22 (R2 = 0.9932) (2)
2.8. Characterization of PFD-Loaded PVA-CMP IPN
Microsphere. 2.8.1. Estimation of Percentage Yield. The 2
The predicted R of 0.9496 is in agreement with the adjusted
total amount of microsphere prepared for each formulation R2 of 0.9848 (p ≤ 0.05).
(F1−F9) was calculated in terms of percentage yield, and is where y2 is the drug entrapment efficiency, x1 and x2 are the
shown in Table 3. The yield was found to be dependent on the quantity of GA (mL) and percentage DL, respectively, and its
concentration of GA and percentage drug loading (% DL) in contour plot is shown in Figure S16b, Supporting Information.
the system. The results showed that yield was found to be 2.8.3. Particle Size Analysis. The arithmetic mean diameter
decreased with increasing drug loading due to limited solubility [d (0.5)] was found, and also the polydispersity index of
of PFD (20 mg/mL) and remained unentrapped in the system. microspheres (F1−F9) was found in the range of 810.12−
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Data presented as avg. ± standard error mean (SEM, n = 3) and statistically analyzed by GraphPad Prism (version 5, GraphPad Software, Inc., California Corporation) using two-tailed Student’s t-test
(p < 0.05). bCmax: peak plasma concentration; Tmax: time taken to reach peak plasma concentration; AUC: area under curve; t1/2: elimination half-life; Kel: elimination rate constant; AUMC: area under
3.23 ± 0.01
4.60** ± 0.24
MRT (h)
gifted sample from the principle manufacturer Hayashibara,
Japan, supplied by Gangwal Chemicals, Mumbai, India.
Poly(vinyl alcohol) (PVA) 88% hydrolyzed (from Thermo
Fisher Scientific, Mumbai, India), light liquid paraffin (from
HiMedia Laboratories Pvt. Ltd., Mumbai, India), Tween-80,
hydrochloric acid (HCl), glycine, petroleum ether (from
Rankem, Gurgaon, Haryana), glutaraldehyde (GA) (from
AUMC (ng h2/mL)
32 945** ± 4200
13 040 ± 370
7103** ± 540
of nitrogen gas at 20 mL/min. The activation energy (Ea) was MTT compared to control assigning 100% viability. Data were
calculated using two different isoconversional kinetic models, reported as mean ± standard deviation (SD) of three different
i.e., Ozawa−Flynn−Wall (OFW)60,61 and Kissinger−Akahira− independent observations.
Sunose (KAS)62,63 for different conversion factors (α) from 4.7.2. In Vitro Biocompatibility Test. Biocompatibility of
0.1 to 0.9, using eqs 8 and 9, respectively, and the detailed CMP and its microsphere was accessed by visualizing cellular
discussion is provided in Section S.4.5, Supporting Informa- morphology using cell imaging technique with fluorescent dyes
ÅÄÅ i ÑÉÑ i
tion. and FESEM analysis.
ÅÅ j Aα R zy ÑÑ j E zy
Å
ln(βi ) = ÅÅlnjj j z
z − ln f (α)ÑÑÑ − jjj ai zzz
4.7.2.1. Cell Imaging. To visualize cellular morphology,
ÅÅ j Eai zz ÑÑ j RTαi z
cells were stained with rhodamine-phalloidin fluorescent dye
ÅÇ k { ÑÖ k {
(Invitrogen) following the previously described protocol.67
Ä ÉÑ
ij βi yz ÅÅÅÅ ij Aα R yz
(8)
ÑÑ i E y
Briefly, prior to staining, cells of 3 days culture were fixed in
j z
lnjj 2 zz = ÅÅÅlnjjj Ñ j z
zzz − ln f (α)ÑÑÑ − jjj αi zzz
10% formalin solution (Sigma) overnight at room temperature.
j T z ÅÅ j E z Ñ
Ñ j z
k αi { ÅÇ k αi { ÑÖ k αi {
The fixed cells were then washed in PBS and incubated with
RT (9) 1% bovine serum albumin (BSA, Sigma) for 30 min to inhibit
the nonspecific binding of dye. This was further followed by
4.6. In Vitro Enzymatic Degradation Study. In vitro PBS washing and permeabilization with 0.1% Triton-X-100
degradation study was conducted using enzyme pullulanase (Sigma) for 5 min. After thorough PBS washing, the cells were
(Sigma, St. Louis, E.C. No. 3.2.1.41). Both the test substances, stained with rhodamine-phalloidin (0.165 μM) for 20 min in
CMP and microsphere, were incubated in a water bath at 37 the dark. To visualize the nucleus, the cells were counter-
°C, and an aliquot of 1 mL of sample was withdrawn at stained with 1 μg/mL Hoechst 33342 (Sigma) for 30 min. The
predetermined time intervals. These samples were analyzed by images were recorded using a fluorescent microscope (EVOS
reducing sugar assay method64,65 as described in Section S.4.6, XL digital, Invitrogen).
Supporting Information. 4.7.2.2. Field Emission Scanning Electron Microscopy
4.6.1. Predictive Modeling for Digestibility. The degrada- (FESEM) Study. FESEM study was performed for the cell-
tion data were best fitted to first-order equation, and its first- seeded microspheres of 3 days culture. These cells were fixed
order derivative yielded logarithm of slope (LOS).43,44 in 2.5% GA for 3 h at room temperature, followed by gradual
Different parameters like digestibility rate constant (k) and dehydration with ethanol (30−100% v/v) and subsequently
the concentration of the product at the end point of reaction allowed to dry completely in vacuum condition. The samples
(C∞) were calculated from LOS plot using the following were then mounted on an aluminum stub, followed by gold
equations sputter coating for 130 s before analysis. The images were
recorded by a field emission electron microscope (Zeiss,
Ct = C∞(1 − e−kt ) (10)
i dC y
Sigma, Germany) at an operating voltage of 2 kV with a
k dt {
4.8. Acute Oral Toxicity Study. Acute oral toxicity studies
(11) of CMP and PVA-CMP placebo IPN microspheres were
where Ct is the product concentration at time (t), C∞ is the conducted as per Organization of Economic Co-operation and
product concentration at the end of the reaction, k is the Development guideline for the test of chemicals 425, adopted
on 17 December 2001. Details of the experimental procedure
( ddCt ) is the logarithm of slope.
digestibility rate constant, and ln are provided in Section S.4.8, Supporting Information.
4.7. In Vitro Cytotoxicity Test and Biocompatibility 4.9. Preparation of PFD-Loaded PVA-CMP IPN Micro-
Assessment. 4.7.1. Cell Viability Study. Cell viability study spheres. The drug-loaded microspheres were prepared by
was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5- adding required amount of PFD to the aqueous phase, and the
diphenyltetrazolium bromide (MTT) (Sigma) assay, as remaining process was similar to that described in Section 4.3.
described earlier.66 Cell culture and maintenance are discussed 4.9.1. Formulation Variables and Factorial Design. In the
in Section S.4.7.1, Supporting Information. HepG2 cells (180 present study, 32 full factorial design was adopted and a total of
μL) were seeded in a 96-well flat-bottomed plate at a nine formulations were prepared using the two factors
concentration of 1 × 104 cells/well and incubated at 37 °C [quantity (mL) of cross-linking agent (X1) and % (w/w)
in a humidified atmosphere of 5% CO2 for 24 h. Thereafter, drug loading ratio (X2)] at three different levels [high level
the cells were treated with 20 μL of various concentrations (+1), medium level (0), and low level (−1)]. The percentage
(1000−50 μg/mL) of CMP (previously sterilized by UV light yield (Y1), drug entrapment efficiency (Y2), cumulative
for three cycles 30 min each, and dissolved in incomplete percentage drug release in 0.1 N HCl after 2 h (Y3) and in
medium) for 72 h. For microspheres, 1 × 104 cells were seeded phosphate buffer after 8 h (Y4) were selected as the responses.
onto preconditioned samples leaving in undisturbed condition Mathematical equation model was generated by 32 factorial
to allow cells adherence on them. Control wells did not design,68 using Design-Expert software (trial version 7.0.0,
contain any test sample except 20 μL of phosphate-buffered Stat-Ease, Inc., Minneapolis) to analyze the generated
saline (PBS). At predetermined time frame, spent media was responses from dependent variables.
replaced by fresh media (180 μL) and aliquot of 20 μL of
MTT from stock (5 mg/mL) solution was added to each well. y = b0 + b1x1 + b2x 2 + b12x1x 2 + b11x12 + b22x 22 (12)
The plate was then incubated at 37 °C for 3 h and then
replaced by 200 μL of dimethyl sulfoxide to solubilize The different formulations according to the factorial design are
formazan crystal. Absorbance was taken at 570 nm using a presented in Table S4, Supporting Information, and the
multiplate reader (Tecan Infinite 200 PRO series, Switzer- contour plot of responses is presented in Figure S16,
land). Cell viability was measured as percentage reduction of Supporting Information.
12006 DOI: 10.1021/acsomega.8b00803
ACS Omega 2018, 3, 11993−12009
ACS Omega
■
Article
■
microsphere optimized formulation (F5) using high-perform-
ance liquid chromatography (HPLC, Waters 1525, Massachu- AUTHOR INFORMATION
setts), with a slight modification in the analytical method as
previously reported.71 Briefly, analysis was performed using Corresponding Authors
C18 column, 250 mm × 4.6 mm, particle size 5 μm (Thermo *E-mail: aghosh@bitmesra.ac.in, anim_1607@yahoo.co.in
(A.G.).
Fisher Scientific, Massachusetts), via isocratic elution mode
*E-mail: biman.mandal@iitg.ac.in, mandal.biman@gmail.com
with a mobile phase of acetonitrile/water containing 0.1% (B.B.M.).
trifluoroacetic acid at a ratio of 35:65 (% v/v) with a flow rate
ORCID
of 1 mL/min using a photodiode array detector at 310 nm.
Liquid−liquid extraction technique (ethyl acetate as extractive Biman B. Mandal: 0000-0003-3936-4621
solvent) was adopted for the extraction of analytes from Animesh Ghosh: 0000-0002-2990-4738
biological matrix. The calibration curve of PFD in plasma was Notes
The authors declare no competing financial interest.
■
plotted in the range of 100−3000 ng/mL with tinidazole (1500
ng/mL) as internal standard. The run time for each analysis
was fixed at 30 min and data acquisition was performed using ACKNOWLEDGMENTS
Breeze software. A.G. sincerely acknowledges the financial support of SERB,
4.11.2. Pharmacokinetic Analysis. A single-dose pharma- New Delhi, through an EMR Individual Centric grant
cokinetic study was conducted on white albino rabbits with a (File EMR/2014/000099). A.G. sincerely acknowledges
PFD equivalent dose of 15 mg/kg body weight. The rabbits support of ACS in form of ACS Author Reward
were classified into two groups (n = 3), group I (dosed with (ARHD3C2R0F0D9K312QX9) which has been redeemed to
Pirfenex) and group II (dosed with F5), using oral gastric purchase an open access license (Author Choice) for this
intubation tube. Blood samples (1 mL) were collected at article. The authors are grateful to Biophore India
Pharmaceuticals Private Limited for the gift sample of PFD,
predetermined time intervals from marginal ear vein at 0 h
and Gangwal Chemicals Private Limited, India, for supplying
(predose) and at 0.5, 1, 1.5, 2, 3, 4, 6, 9, and 12 h (postdose) in
the gift sample of pullulan from the principle manufacturer
K3-EDTA vials (Peerless Biotech Pvt. Ltd., Chennai, Tamil Hayashibara Co. Ltd., Japan. B.B.M. greatly acknowledges
Nadu, India). The blood samples were centrifuged for 10 min, funding support from Department of Biotechnology (DBT)
and at −4 °C, plasma was separated and analyzed using and Department of Science and Technology (DST), Govt. of
validated HPLC method as earlier described (Section 4.11.1). India. The authors acknowledge Biomaterial and Tissue
The pharmacokinetic parameters were calculated using zero Engineering Laboratory, Department of Biosciences and
moment noncompartment pharmacokinetic modeling.72 The Bioengineering, IIT Guwahati, for providing necessary facilities
procedure followed for animal housing and handling is for conducting cytocompatibility studies and Department of
provided in Section S.4.11.2, Supporting Information. Pharmaceutical Sciences and Technology along with CIF
12007 DOI: 10.1021/acsomega.8b00803
ACS Omega 2018, 3, 11993−12009
ACS Omega Article
facility of BIT Mesra, Ranchi, for providing all other required (19) Rokhade, A. P.; Agnihotri, S. A.; Patil, S. A.; Mallikarjuna, N.
facilities. N.; Kulkarni, P. V.; Aminabhavi, T. M. Semi-interpenetrating polymer
■
network microspheres of gelatin and sodium carboxymethyl cellulose
ABBREVIATIONS for controlled release of ketorolac tromethamine. Carbohydr. Polym.
2006, 65, 243−252.
PVA, poly(vinyl alcohol); GA, glutaraldehyde; PFD, pirfeni- (20) Matricardi, P.; Di Meo, C.; Coviello, T.; Hennink, W. E.;
done; CMP, carboxymethyl pullulan; IPN, interpenetrating Alhaique, F. Interpenetrating polymer networks polysaccharide
polymeric network; IPF, idiopathic pulmonary fibrosis; MTT, hydrogels for drug delivery and tissue engineering. Adv. Drug Delivery
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Rev. 2013, 65, 1172−1187.
■ REFERENCES
(1) Dubey, R. D.; Saneja, A.; Gupta, P. K.; Gupta, P. N. Recent
(21) Soni, S. R.; Ghosh, A. Exploring pullulan-poly(vinyl alcohol)
interpenetrating network microspheres as controlled release drug
delivery device. Carbohydr. Polym. 2017, 174, 812−822.
advances in drug delivery strategies for improved therapeutic efficacy (22) Shaw, J.; Marshall, T.; Morris, H.; Hayton, C.; Chaudhuri, N.
of gemcitabine. Eur. J. Pharm. Sci. 2016, 93, 147−162. Idiopathic pulmonary fibrosis: a holistic approach to disease
(2) Future Market Insights (FMI) Report, 2017. https://www. management in the antifibrotic age. J. Thorac. Dis. 2017, 9, 4700.
prnewswire.com/news-releases/solid-dosage-forms-will-remain- (23) Pirfenidone. USFDA Reviews. Pharmacology Review(s); Center
preferred-in-the-global-oral-controlled-release-drug-delivery- for Drug Evaluation and Research, 2014; pp 1−496.
technology-market-637852753.html (last assessed May 02, 2018). (24) Jana, S.; Sharma, R.; Maiti, S.; Sen, K. K. Interpenetrating
(3) Santos, L. F.; Correia, I. J.; Silva, A. S.; Mano, J. F. Biomaterials hydrogels of O-carboxymethyl Tamarind gum and alginate for
for drug delivery patches. Eur. J. Pharm. Sci. 2018, 118, 49−66. monitoring delivery of acyclovir. Int. J. Biol. Macromol. 2016, 92,
(4) Degen, L.; Phillips, S. Variability of gastrointestinal transit in 1034−1039.
healthy women and men. Gut 1996, 39, 299−305. (25) Kaity, S.; Ghosh, A. Carboxymethylation of locust bean gum:
(5) Gupta, U.; Perumal, O. Dendrimers and Its Biomedical application in interpenetrating polymer network microspheres for
Applications. In Natural and Synthetic Biomedical Polymers; Elsevier, controlled drug delivery. Ind. Eng. Chem. Res. 2013, 52, 10033−
2014; pp 243−257. 10045.
(6) Ahsan, S. M.; Thomas, M.; Reddy, K. K.; Sooraparaju, S. G.; (26) Pushpamalar, V.; Langford, S.; Ahmad, M.; Lim, Y.
Asthana, A.; Bhatnagar, I. Chitosan as biomaterial in drug delivery and Optimization of reaction conditions for preparing carboxymethyl
tissue engineering. Int. J. Biol. Macromol. 2018, 110, 97−109. cellulose from sago waste. Carbohydr. Polym. 2006, 64, 312−318.
(7) Kumar, A.; Rao, K. M.; Han, S. S. Application of xanthan gum as (27) Sharma, B. R.; Kumar, V.; Soni, P. Carbamoylethylation of
polysaccharide in tissue engineering: A review. Carbohydr. Polym. Cassia tora gum. Carbohydr. Polym. 2003, 54, 143−147.
2018, 180, 128−144. (28) Aminabhavi, T. M.; Nadagouda, M. N.; More, U. A.; Joshi, S.
(8) Ali, A.; Ahmed, S. A Review on Chitosan and its Nano- D.; Kulkarni, V. H.; Noolvi, M. N.; Kulkarni, P. V. Controlled release
composites in Drug Delivery. Int. J. Biol. Macromol. 2018, 109, 273− of therapeutics using interpenetrating polymeric networks. Expert
286. Opin. Drug Delivery 2015, 12, 669−688.
(9) Vijan, V.; Kaity, S.; Biswas, S.; Isaac, J.; Ghosh, A. Microwave (29) Whistler, R. L.; BeMiller, J. Alkaline Degradation of
assisted synthesis and characterization of acrylamide grafted gellan, Polysaccharides. Advances in Carbohydrate Chemistry; Elsevier, 1958;
application in drug delivery. Carbohydr. Polym. 2012, 90, 496−506. pp 289−329.
(10) Kaity, S.; Isaac, J.; Kumar, P. M.; Bose, A.; Wong, T. W.; (30) Alekhina, M.; Mikkonen, K. S.; Alén, R.; Tenkanen, M.; Sixta,
Ghosh, A. Microwave assisted synthesis of acrylamide grafted locust H. Carboxymethylation of alkali extracted xylan for preparation of
bean gum and its application in drug delivery. Carbohydr. Polym. bio-based packaging films. Carbohydr. Polym. 2014, 100, 89−96.
2013, 98, 1083−1094. (31) Yuen, S.-N.; Choi, S.-M.; Phillips, D. L.; Ma, C.-Y. Raman and
(11) Prabaharan, M. Prospective of guar gum and its derivatives as FTIR spectroscopic study of carboxymethylated non-starch poly-
controlled drug delivery systems. Int. J. Biol. Macromol. 2011, 49, saccharides. Food Chem. 2009, 114, 1091−1098.
117−124. (32) Mansur, H. S.; Oréfice, R. L.; Mansur, A. A. Characterization of
(12) Al-Karawi, A. J. M.; Al-Daraji, A. H. R. Preparation and using of poly(vinyl alcohol)/poly(ethylene glycol) hydrogels and PVA-derived
acrylamide grafted starch as polymer drug carrier. Carbohydr. Polym.
hybrids by small-angle X-ray scattering and FTIR spectroscopy.
2010, 79, 769−774.
Polymer 2004, 45, 7193−7202.
(13) Shao, Y.; Li, L.; Gu, X.; Wang, L.; Mao, S. Evaluation of
(33) Badr, Y. A.; El-Kader, K. M. A.; Khafagy, R. M. Raman
chitosan−anionic polymers based tablets for extended-release of
spectroscopic study of CdS, PVA composite films. J. Appl. Polym. Sci.
highly water-soluble drugs. Asian J. Pharm. Sci. 2015, 10, 24−30.
(14) Viridén, A.; Larsson, A.; Schagerlöf, H.; Wittgren, B. Model 2004, 92, 1984−1992.
drug release from matrix tablets composed of HPMC with different (34) Masuda, K.; Kaji, H.; Horii, F. CP/MAS 13C NMR analyses of
substituent heterogeneity. Int. J. Pharm. 2010, 401, 60−67. hydrogen bonding and the chain conformation in the crystalline and
(15) Mittermayr, R.; Slezak, P.; Haffner, N.; Smolen, D.; Hartinger, noncrystalline regions for poly(vinyl alcohol) films. J. Polym. Sci., Part
J.; Hofmann, A.; Schense, J.; Spazierer, D.; Gampfer, J.; Goppelt, A.; B: Polym. Phys. 2000, 38, 1−9.
Redl, H. Controlled release of fibrin matrix-conjugated platelet (35) Yang, H.; Hu, S.; Horii, F.; Endo, R.; Hayashi, T. CP/MAS 13C
derived growth factor improves ischemic tissue regeneration by NMR analysis of the structure and hydrogen bonding of melt-
functional angiogenesis. Acta Biomater. 2016, 29, 11−20. crystallized poly(vinyl alcohol) films. Polymer 2006, 47, 1995−2000.
(16) Gander, B.; Gurny, R.; Doelker, E.; Peppas, N. A. Effect of (36) Kajjari, P. B.; Manjeshwar, L. S.; Aminabhavi, T. M. Semi-
polymeric network structure on drug release from cross-linked interpenetrating polymer network hydrogel blend microspheres of
poly(vinyl alcohol) micromatrices. Pharm. Res. 1989, 6, 578−584. gelatin and hydroxyethyl cellulose for controlled release of theophyl-
(17) Martinez, A. W.; Caves, J. M.; Ravi, S.; Li, W.; Chaikof, E. L. line. Ind. Eng. Chem. Res. 2011, 50, 7833−7840.
Effects of crosslinking on the mechanical properties, drug release and (37) Kajjari, P. B.; Manjeshwar, L. S.; Aminabhavi, T. M. Novel
cytocompatibility of protein polymers. Acta Biomater. 2014, 10, 26− interpenetrating polymer network hydrogel microspheres of chitosan
33. and poly(acrylamide)-grafted-guar gum for controlled release of
(18) Carbinatto, F. M.; de Castro, A. D.; Evangelista, R. C.; Cury, B. ciprofloxacin. Ind. Eng. Chem. Res. 2011, 50, 13280−13287.
S. Insights into the swelling process and drug release mechanisms (38) Park, J. W.; Oh, S. C.; Lee, H. P.; Kim, H. T.; Yoo, K. O. A
from cross-linked pectin/high amylose starch matrices. Asian J. Pharm. kinetic analysis of thermal degradation of polymers using a dynamic
Sci. 2014, 9, 27−34. method. Polym. Degrad. Stab. 2000, 67, 535−540.
(39) Jakab, E.; Faix, O.; Till, F.; Székely, T. The effect of cations on (58) Eyler, R.; Klug, E.; Diephuis, F. Determination of degree of
the thermal decomposition of lignins. J. Anal. Appl. Pyrolysis 1993, 25, substitution of sodium carboxymethylcellulose. Anal. Chem. 1947, 19,
185−194. 24−27.
(40) Gómez, I.; Otazo, E. M.; Hernández, H.; Rubio, E.; Varela, J.; (59) Kaity, S.; Ghosh, A. Facile preparation of acrylamide grafted
Ramírez, M.; Barajas, I.; Gordillo, A. J. Thermal degradation study of locust bean gum-poly(vinyl alcohol) interpenetrating polymer net-
PVA derivative with pendant phenylthionecarbamate groups by DSC/ work microspheres for controlled oral drug delivery. J. Drug Delivery
TGA and GC/MS. Polym. Degrad. Stab. 2015, 112, 132−136. Sci. Technol. 2016, 33, 1−12.
(41) Chrissafis, K.; Paraskevopoulos, K.; Bikiaris, D. Thermal (60) Flynn, J. H.; Wall, L. A. General treatment of the
degradation kinetics of the biodegradable aliphatic polyester, thermogravimetry of polymers. J. Res. Natl. Bur. Stand., Sect. A
poly(propylene succinate). Polym. Degrad. Stab. 2006, 91, 60−68. 1966, 70A, 487−523.
(42) Wu, S.-J.; Chen, J. Preparation of maltotriose from (61) Ozawa, T. A new method of analyzing thermogravimetric data.
fermentation broth by hydrolysis of pullulan using pullulanase. Bull. Chem. Soc. Jpn. 1965, 38, 1881−1886.
(62) Akahira, T.; Sunose, T. Method of Determining Activation
Carbohydr. Polym. 2014, 107, 94−97.
Deterioration Constant of Electrical Insulating; Research Report;
(43) Edwards, C. H.; Warren, F. J.; Milligan, P. J.; Butterworth, P. J.;
Institute of Technology, 1971; Vol. 16, pp 22−31.
Ellis, P. R. A novel method for classifying starch digestion by
(63) Kissinger, H. E. Reaction kinetics in differential thermal
modelling the amylolysis of plant foods using first-order enzyme analysis. Anal. Chem. 1957, 29, 1702−1706.
kinetic principles. Food Funct. 2014, 5, 2751−2758. (64) Ali, G.; Rihouey, C.; Le Cerf, D.; Picton, L. Effect of
(44) Butterworth, P. J.; Warren, F. J.; Grassby, T.; Patel, H.; Ellis, P. carboxymethyl groups on degradation of modified pullulan by
R. Analysis of starch amylolysis using plots for first-order kinetics. pullulanase from Klebsiella pneumoniae. Carbohydr. Polym. 2013, 93,
Carbohydr. Polym. 2012, 87, 2189−2197. 109−115.
(45) Reddy, K. M.; Babu, V. R.; Sairam, M.; Subha, M. C. S.; (65) Saqib, A. A. N.; Whitney, P. J. Differential behaviour of the
Mallikarjuna, N. N.; Kulkarni, P. V.; Aminabhavi, T. M. Development dinitrosalicylic acid (DNS) reagent towards mono-and di-saccharide
of chitosan-guar gum semi-interpenetrating polymer network micro- sugars. Biomass Bioenergy 2011, 35, 4748−4750.
spheres for controlled release of cefadroxil. Des. Monomers Polym. (66) Kumar, J. P.; Mandal, B. B. Antioxidant potential of mulberry
2006, 9, 491−501. and non-mulberry silk sericin and its implications in biomedicine. Free
(46) Das, D.; Das, R.; Ghosh, P.; Dhara, S.; Panda, A. B.; Pal, S. Radic. Biol. Med. 2017, 108, 803−818.
Dextrin cross linked with poly(HEMA): a novel hydrogel for colon (67) Joseph, C. M.; Reardon, P. J. T.; Konwarh, R.; Knowles, J. C.;
specific delivery of ornidazole. RSC Adv. 2013, 3, 25340−25350. Mandal, B. B. Mimicking Hierarchical Complexity of the Osteochon-
(47) Angadi, S. C.; Manjeshwar, L. S.; Aminabhavi, T. M. Coated dral Interface Using Electrospun Silk-Bioactive Glass Composites.
interpenetrating blend microparticles of chitosan and guar gum for ACS Appl. Mater. Interfaces 2017, 9, 8000−8013.
controlled release of isoniazid. Ind. Eng. Chem. Res. 2013, 52, 6399− (68) Kumar, P. M.; Ghosh, A. Development and evaluation of silver
6409. sulfadiazine loaded microsponge based gel for partial thickness
(48) Babu, V. R.; Sairam, M.; Hosamani, K.; Aminabhavi, T. (second degree) burn wounds. Eur. J. Pharm. Sci. 2017, 96, 243−254.
Preparation and characterization of novel semi-interpenetrating 2- (69) Kaity, S.; Isaac, J.; Ghosh, A. Interpenetrating polymer network
hydroxyethyl methacrylate-g-chitosan copolymeric microspheres for of locust bean gum-poly(vinyl alcohol) for controlled release drug
sustained release of indomethacin. J. Appl. Polym. Sci. 2007, 106, delivery. Carbohydr. Polym. 2013, 94, 456−467.
(70) Isaac, J.; Kaity, S.; Ganguly, S.; Ghosh, A. Microwave-induced
3778−3785.
solid dispersion technology to improve bioavailability of glipizide. J.
(49) Rokhade, A. P.; Kulkarni, P. V.; Mallikarjuna, N. N.;
Pharm. Pharmacol. 2013, 65, 219−229.
Aminabhavi, T. M. Preparation and characterization of novel semi-
(71) Shi, S.; Wu, J.; Chen, H.; Chen, H.; Wu, J.; Zeng, F. Single-and
interpenetrating polymer network hydrogel microspheres of chitosan Multiple-Dose Pharmacokinetics of Pirfenidone, an Antifibrotic
and hydroxypropyl cellulose for controlled release of chlorothiazide. J. Agent, in Healthy Chinese Volunteers. J. Clin. Pharmacol. 2007, 47,
Microencapsulation 2009, 26, 27−36. 1268−1276.
(50) Reddy, K. M.; Babu, V. R.; Rao, K. S. V. K.; Subha, M. C. S.; (72) Kaity, S.; Ghosh, A. Comparative bio-safety and in vivo
Rao, K. C.; Sairam, M.; Aminabhavi, T. M. Temperature sensitive evaluation of native or modified locust bean gum-PVA IPN
semi-IPN microspheres from sodium alginate and N-isopropylacry- microspheres. Int. J. Biol. Macromol. 2015, 72, 883−893.
lamide for controlled release of 5-fluorouracil. J. Appl. Polym. Sci.
2008, 107, 2820−2829.
(51) Varelas, C. G.; Dixon, D. G.; Steiner, C. A. Zero-order release
from biphasic polymer hydrogels. J. Controlled Release 1995, 34, 185−
192.
(52) Wagner, J. G. Interpretation of percent dissolved-time plots
derived from in vitro testing of conventional tablets and capsules. J.
Pharm. Sci. 1969, 58, 1253−1257.
(53) Costa, P.; Lobo, J. M. S. Modeling and comparison of
dissolution profiles. Eur. J. Pharm. Sci. 2001, 13, 123−133.
(54) Mulye, N.; Turco, S. A simple model based on first order
kinetics to explain release of highly water soluble drugs from porous
dicalcium phosphate dihydrate matrices. Drug Dev. Ind. Pharm. 1995,
21, 943−953.
(55) Hixson, A.; Crowell, J. Dependence of reaction velocity upon
surface and agitation. Ind. Eng. Chem. 1931, 23, 1160−1168.
(56) Higuchi, T. Mechanism of sustained-action medication.
Theoretical analysis of rate of release of solid drugs dispersed in
solid matrices. J. Pharm. Sci. 1963, 52, 1145−1149.
(57) Korsmeyer, R. W.; Gurny, R.; Doelker, E.; Buri, P.; Peppas, N.
A. Mechanisms of solute release from porous hydrophilic polymers.
Int. J. Pharm. 1983, 15, 25−35.