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Optimization and Formulation of Fucoxanthin-Loaded Microsphere (F-LM) Using Response Surface Methodology (RSM) and Analysis of Its Fucoxanthin Release Profile

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Fucoxanthin has interesting anticancer activity, but is insoluble in water, hindering its use as a drug. Microencapsulation is used as a technique for improving drug delivery. This study aimed to formulate fucoxanthin-loaded microspheres (F-LM) for anticancer treatment of H1299 cancer cell lines and optimize particle size (PS) and encapsulation efficiency (EE). Using response surface methodology (RSM), a face centered central composite design (FCCCD) was designed with three factors: Polyvinylalcohol (PVA), poly(d,l-lactic-co-glycolic acid) (PLGA), and fucoxanthin concentration. F-LM was produced using a modified double-emulsion solvent evaporation method. The F-LM were characterized for release profile, release kinetics, and degradation pattern. Optimal F-LM PS and EE of 9.18 µm and 33.09%, respectively, with good surface morphology, were achieved from a 0.5% (w/v) PVA, 6.0% (w/v) PLGA, 200 µg/mL fucoxanthin formulation at a homogenization speed of 20,500 rpm. PVA concentration was the most significant factor (p < 0.05) affecting PS. Meanwhile, EE was significantly affected by interaction between the three factors: PVA, PLGA, and fucoxanthin. In vitro release curve showed fucoxanthin had a high burst release (38.3%) at the first hour, followed by a sustained release stage reaching (79.1%) within 2 months. Release kinetics followed a diffusion pattern predominantly controlled by the Higuchi model. Biodegradability studies based on surface morphology changes on the surface of the F-LM, show that morphology changed within the first hour, and F-LM completely degraded within 2 months. RSM under FCCCD design improved the difference between the lowest and highest responses, with good correlation between observed and predicted values for PS and EE of F-LM.
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molecules
Article
Optimization and Formulation of
Fucoxanthin-Loaded Microsphere (F-LM) Using
Response Surface Methodology (RSM) and Analysis
of Its Fucoxanthin Release Profile
Irwandi Jaswir 1,2,3,*, Dedi Noviendri 2, Muhammad Taher 4, Farahidah Mohamed 4,
Fitri Octavianti 5, Widya Lestari 1, Ali Ghufron Mukti 6, Sapta Nirwandar 7and
Bubaker B. Hamad Almansori 1
1
International Institute for Halal Research and Training (INHART), International Islamic University Malaysia,
Jalan Gombak, Kuala Lumpur 53100, Malaysia; dokterwidya@gmail.com (W.L.);
science.abu@gmail.com (B.B.H.A.)
2Bioprocess and Molecular Engineering Research Unit (BPMERU), International Islamic University
Malaysia (IIUM) Gombak, Kuala Lumpur 53100, Malaysia; ir98@hotmail.com
3Department of Pharmaceutical Technology, Universitas Ahmad Dahlan, Yogyakarta 55164, Indonesia
4
Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia
Kuantan, Kuantan 25200, Malaysia; mtaher@iium.edu.my (M.T.); farahidah@iium.edu.my (F.M.)
5
Department of Orthodontics, Faculty of Dentistry, Universiti Sains Islam Malaysia, Tower B, Persiaran MPAJ,
Jalan Pandan Utama, Kuala Lumpur 55100, Malaysia; yanti75@yahoo.com
6Ministry of Research, Technology and Higher Education of Indonesia, Senayan, Jakarta Pusat 10340,
Indonesia; ghufronmukti@yahoo.com
7Chairman, Indonesia Halal Lifestyle Foundation, Jakarta 10230, Indonesia; bob.naedi@gmail.com
*Correspondence: irwandi@iium.edu.my; Tel.: +603-619-66-498
Academic Editor: Benoît Schoefs
Received: 3 January 2019; Accepted: 27 February 2019; Published: 7 March 2019


Abstract:
Fucoxanthin has interesting anticancer activity, but is insoluble in water, hindering its
use as a drug. Microencapsulation is used as a technique for improving drug delivery. This study
aimed to formulate fucoxanthin-loaded microspheres (F-LM) for anticancer treatment of H1299
cancer cell lines and optimize particle size (PS) and encapsulation efficiency (EE). Using response
surface methodology (RSM), a face centered central composite design (FCCCD) was designed with
three factors: Polyvinylalcohol (PVA), poly(D,L-lactic-co-glycolic acid) (PLGA), and fucoxanthin
concentration. F-LM was produced using a modified double-emulsion solvent evaporation method.
The F-LM were characterized for release profile, release kinetics, and degradation pattern. Optimal
F-LM PS and EE of 9.18
µ
m and 33.09%, respectively, with good surface morphology, were achieved
from a 0.5% (w/v) PVA, 6.0% (w/v) PLGA, 200
µ
g/mL fucoxanthin formulation at a homogenization
speed of 20,500 rpm. PVA concentration was the most significant factor (p< 0.05) affecting PS.
Meanwhile, EE was significantly affected by interaction between the three factors: PVA, PLGA,
and fucoxanthin.
In vitro
release curve showed fucoxanthin had a high burst release (38.3%) at the
first hour, followed by a sustained release stage reaching (79.1%) within 2 months. Release kinetics
followed a diffusion pattern predominantly controlled by the Higuchi model. Biodegradability
studies based on surface morphology changes on the surface of the F-LM, show that morphology
changed within the first hour, and F-LM completely degraded within 2 months. RSM under FCCCD
design improved the difference between the lowest and highest responses, with good correlation
between observed and predicted values for PS and EE of F-LM.
Keywords:
fucoxanthin; release profile; response surface methodology; microsphere;
microencapsulation
Molecules 2019,24, 947; doi:10.3390/molecules24050947 www.mdpi.com/journal/molecules
Molecules 2019,24, 947 2 of 16
1. Introduction
Fucoxanthin, a major xanthophylls in brown seaweed, has a unique structure, including a usual
allenic bond and 5,6-monoepoxide in its molecule. Fucoxanthin has been reported to induce apoptosis
in prostate cancer PC-3, DU145 and LNCap cells, leukemia HL-60 cells, and caused cell cycle arrest
during the G
0
/G
1
phase in neuroblastoma GOTO cells [
1
] Furthermore, in colon cancer cell lines,
fucoxanthin has been shown to induces apoptosis in Caco-2, HT-29 and DLD-1 cells [14].
Health care systems is Japan, Korea, and India use fucoxanthin for many applications, such
as anticancer, antiobesity, antidiabetic, to induce cell cycle arrest at G
0
/G
1
phase in human colon
carcinoma cells, antioxidant, and anti-inflammatory applications. This compound can be isolated from
varying types of brown seaweed, such as Undaria pinnatifida,Hijkia fusiformis,Sargassum fulvellum, and
Laminaria japonica, from Japan, Padina tetrastomatica from India, and Sargassum siliquastrum from Korea,
as well as T. turbinata and S. plagyophyllum from the Malaysian Peninsular [2,57].
Fucoxanthin has the potential to become an anticancer drug candidate due to its anticancer
activities. Unfortunately, this carotenoid is insoluble in water, which poses a problem for its use as
a drug candidate. Microencapsulation (ME) is one of the most interesting drug delivery systems [
1
],
which can delay and modify drug release from pharmaceutical dosage forms [
2
]. Many studies have
reported the advantages of formulation in the delivery of insoluble drugs using ME [36].
RSM is the collection of statistical techniques useful for developing, improving, and optimizing
processes [
8
]. RSM defines the effect of the independent variables, alone or in combination, on the
process [
9
]. The main advantage of RSM is to reduce the number of experimental trials needed to
evaluate multiple variables [
10
] and its interactions; it is less laborious and time-consuming than other
approaches [11,12].
The objective of this study was to optimize and formulate fucoxanthin-loaded microspheres
(F-LM) for anticancer treatment of the human lung cancer (H1299) cell line using response surface
methodology (RSM). Particle size (PS) and encapsulation efficiency (EE) of the F-LM fabricated via
microencapsulation technique and fucoxanthin release profile, release kinetics, and its degradation.
2. Results and Discussion
2.1. Optimization of Microencapsulation Component by RSM
A face centered central composite design (FCCCD) under RSM was used to investigate the optimal
conditions of the three significant factors (PVA, PLGA, and fucoxanthin concentration) towards PS
and EE for the anticancer treatment on the human lung cancer (H1299) cell line. For each run, the
experimental (observed) results, along with the predicted PS and EE obtained from the regression
equations for the 15 combinations, are shown in Table 1.
The results demonstrated that optimal PS and EE, by double emulsion extraction/evaporation
method (9.18
µ
m and 33.09%), were observed in the runs representing the center point (run 12). This
size is desirable for pulmonary drug delivery, namely between 1 to 10
µ
m. Furthermore, the highest
amounts of PS and EE (10.95
µ
m and 34.87%, respectively), by this method, were observed in the run
representing the center point (run 11) and the lowest amounts were observed in run 15 (2.01
µ
m and
10.25%), where factors such as PLGA and fucoxanthin were at the lowest concentrations, whereas the
PVA concentration was at the highest concentration. In this study, Table 2shows the design matrix of
FCCCD, which further improved the PS and EE, such that the difference between the lowest and the
highest response was (2.01 to 10.95 µm; 10.25% to 34.87%).
Molecules 2019,24, 947 3 of 16
Table 1.
A face centered central composite design (FCCCD) of three independent variables with their
coded and actual values and one center point showing the predicted and experimental response.
Run PVA
(% w/v)
PLGA
(% w/v)Fuco (µg/mL) PS (µm) EE (%)
Ex. Pr.* Ex. Pr.**
1 0.80 (+1) 6.00 (0) 200.00 (0) 5.12 4.38 28.91 28.93
2 0.50 (0) 6.00 (0) 100.00 (1) 7.48 7.90 22.25 22.35
3 0.20 (1) 9.00 (+1) 300.00 (+1) 6.45 6.67 33.97 33.33
4 0.20 (1) 3.00 (1) 100.00 (1) 4.95 4.34 19.85 18.43
5 0.20 (1) 9.00 (+1) 100.00 (1) 5.18 5.22 25.57 26.85
6 0.80 (+1) 9.00 (+1) 100.00 (1) 3.12 3.48 21.15 20.64
7 0.50 (0) 3.00 (1) 200.00 (0) 6.85 8.04 24.56 26.40
8 0.20 (1) 6.00 (0) 200.00 (0) 6.01 6.69 33.94 34.30
9 0.20 (1) 3.00 (1) 300.00 (+1) 5.36 5.02 28.72 29.13
10 0.80 (+1) 9.00 (+1) 300.00 (+1) 3.97 4.59 28.87 30.19
11 0.50 (0) 9.00 (+1) 200.00 (0) 10.95 9.71 34.87 33.41
12 0.50 (0) 6.00 (0) 200.00 (0) 9.18 9.30 33.09 32.34
13 0.80 (+1) 3.00 (1) 300.00 (+1) 2.16 2.13 25.97 24.59
14 0.50 (0) 6.00 (0) 300.00 (+) 9.26 8.79 32.19 32.47
15 0.80 (+1) 3.00 (1) 100.00 (1) 2.01 1.80 10.27 10.82
PVA: Polyvinylalcohol; PLGA: Poly(D,L-lactic-co-glycolic acid); Fuco: Fucoxanthin; PS: Particle size; EE:
Encapsulation efficiency; Ex.: Experiment; Pr.: Predicted. * Second order polynomial (Equaton (2)) was used
to estimate the predicted response (particle size). ** Second order polynomial (Equation (3)) was used to estimate
the predicted response (EE).
A second order regression Equation showed the dependence of PS and EE of F-LM produced
by double emulsion extraction/evaporation method on the microencapsulation components. The
parameters of the equation were obtained by multiple regression analysis of the experimental data [
13
].
An empirical relationship between the screened and response variables were expressed in terms of the
second-order polynomial equation:
Y1(PS, µm) = + 9.30 1.16A+ 0.83B+ 0.45C3.76A20.42B20.95C2+ 0.20AB 0.085AC + 0.20BC (1)
where the PS is the response (Y
1
) and A,B, and Care the concentrations of PVA, PLGA, and
fucoxanthin, respectively.
Y2(EE, %) = +32.34 2.69A+ 3.51B+ 5.06C0.72A22.43B24.93C2+ 0.35AB + 0.77AC 1.06BC (2)
where the EE is the response (Y
2
) and A,B, and Care the concentration of PVA, PLGA, and
fucoxanthin, respectively.
The adequacy of the model for PS and EE were checked using ANOVA. which was tested using
Fisher’s exact test and the results are shown in Tables 2and 3. For PS (Table 2) the model F value of
8.99 and a p-value of <0.0131 imply that the model is significant, suggesting that there is only 1.31%
chance that the model F value could occur due to noise. Model terms with a probability >F (less than
0.05) are considered significant, while those greater than 0.10 are insignificant [
13
]. Furthermore, for
EE (Table 3), the model F value of 23.17 and a p-value of <0.0015 imply that the model is significant,
suggesting that there is only 0.15% chance that the model F value could occur due to noise. The model
terms with a probability >F (less than 0.05) are considered significant [14].
Molecules 2019,24, 947 4 of 16
Table 2. ANOVA of quadratic model for PS (particle size).
Source Sum of Square F-Value p-Value
Model 88.67 8.99 0.0131
PVA, A13.39 12.22 0.0174
PLGA, B6.96 6.35 0.0532
Fuco, C1.99 1.82 0.2357
A236.34 33.17 0.0022
B20.46 0.42 0.5442
C22.34 2.14 0.2036
AB 0.32 0.29 0.6121
AC 0.058 0.054 0.8274
BC 0.30 0.28 0.6208
R2= 0.9418, adjusted R2= 0.8371, adequate precision = 9.249, p< 0.05 was considered to be significant.
Table 3. ANOVA of quadratic model for EE (encapsulation efficiency).
Source Sum of Square F-Value p-Value
Model 614.88 23.17 0.0015
PVA, A72.25 24.51 0.0043
PLGA, B122.92 41.69 0.0013
Fuco, C256.34 86.95 0.0002
A21.35 0.46 0.5287
B215.24 5.71 0.0721
C262.48 21.19 0.0058
AB 0.99 0.33 0.5879
AC 4.73 1.60 0.2612
BC 8.93 3.03 0.1424
R2= 0.9766, adjusted R2= 0.9344, adequate precision = 16.752, p< 0.05 was considered to be significant.
An R
2
value closer to 1 denotes better correlation between the experiment (observed) and
predicted values. For PS (Table 3and Figure 1A), the higher values of R
2
(0.9418) and adjusted
R
2
(0.8371) also indicated the efficacy of the model and 94.18% or 83.71% variations could be accounted
for by the model equation. Thus, for a good statistical model, the R
2
value should be in the range
0 to 1.0, and the closer the value is to 1.0, the better fit the model is deemed to be [
14
]. Moreover,
adequate precision measures signal to noise ratio, and a value >4 is considered a prerequisite for
desirable models [
13
]. The adequate precision value of 9.249 for PS indicates that the model can be
used to navigate the design space. Furthermore, for EE (Table 4and Figure 1B), the higher values of
R
2
(0.9766) and adjusted R
2
(0.9344) also indicated the efficacy of the model, and 97.66% or 93.44%
variations could be accounted for by the model equation. The adequate precision value of 16.752 for
EE indicates that the model can be used to navigate the design space.
Table 4. Regression values of corresponding kinetic equation of F-LM.
Equation Zero Order First Order Higuchi
R20.719 0.792 0.841
Molecules 2019,24, 947 5 of 16
Figure 1.
The design experts plot between predicted and actual values for PS (particle size) (
A
) and EE
(encapsulation efficiency) (B).
The correlation coefficient values of regression equation are listed in Tables 2and 3. The p-value
is used as a tool to check the significance of each coefficient [
14
], which also indicates the interaction
strength between each independent variable. The smaller the p-value, the bigger significance of the
corresponding coefficient. For PS (Table 2), the responses revealed that only one interaction term
(A-PVA), and one the quadratic coefficient (A
2
) were significant (p< 0.05), and had remarkable effects
on the overall PS.
Lakshmana et al. [
15
] and Dhakar et al. [
16
] reported that the PS of microsphere is seen to be
dependent on the concentration of PVA in the continuous phase. From this study, the results revealed
that, with an increase in the concentration of PVA, more PVA molecules may overlay the surface of
the droplets. Increasing of the concentration PVA provides conditions to obtain smaller emulsion
droplets [
17
]. Furthermore, increasing PVA concentration has been shown to provide an increased
protection of the droplets against coalescence resulting in the production of small PS [1719].
For EE (Table 2), the response for one interaction term (A-PVA), (B-PLGA), and (C-fucoxanthin)
and the quadratic coefficient, were significant (p< 0.05) and had remarkable effects on the overall EE.
Ruan and Feng [
20
] reported that the EE is defined as the ratio of amount of encapsulated drug to that
of the drug used for microsphere preparation. Dhakar et al. [
16
] reported that the loading efficiency of
drug release from the microsphere depended on the concentration of polymer and type of polymer
used. EE has been shown to increase alongside an increasing concentration of polymer [
21
]. The high
polymer concentration results in large microspheres, which causes more loss of drug from the surface
during washing of the microspheres compared to smaller microspheres [
16
]. Thus, microsphere size
also affected EE [16].
The 3D response surface plot is the graphical representation of the regression equation used to
investigate the interactions among variables and to determine the optimum concentration of each
factor [
12
] for optimum PS and EE by w/o/wdouble emulsion extraction/evaporation method. The
3D response surface and contour plots of the combined effects of PVA, PLGA, and fucoxanthin
concentration for PS and EE by double emulsion extraction/evaporation method are shown in Figure 2.
The 3D plots are based on the function of concentrations of two variables, with the other variable being
at its optimum level [
9
]. Significant interaction between the corresponding variables is indicated by an
elliptical or saddle nature of the contour plots [
12
,
21
23
]. Figure 2A represents the interaction between
PLGA (coating) and fucoxanthin (core) concentration. Lower and higher levels of both the PLGA and
Molecules 2019,24, 947 6 of 16
fucoxanthin did not result in higher PS. Figure 2B shows the 3D plot corresponding to PVA and PLGA
concentration. In the case of PVA and fucoxanthin (Figure 2C), the response plot was elliptical, showing
interaction between them with optimum PS by double emulsion extraction/evaporation method.
Figure 2.
3D response surface curves and 2D contour of the combined effects of PVA (polyvinylalcohol),
poly lactic-co-glycolic acid (PLGA), and fucoxanthin concentration on PS (particle size) by double
emulsion extraction/evaporation method (
A
) PLGA and fucoxanthin at fixed level of PVA; (
B
) PVA
and PLGA at fixed level of fucoxanthin; (C) PVA and fucoxanthin at fixed level of PLGA.
Furthermore, the elliptical response plot in Figure 2A shows interactions between PLGA
and fucoxanthin with optimum EE by double emulsion extraction/evaporation method, whereas
Molecules 2019,24, 947 7 of 16
Figure 2B,C show the 3D plots corresponding to PVA and PLGA, and PVA and fucoxanthin
concentration, respectively. Lower and higher level PVA and PLGA, and PVA and fucoxanthin
did not result in higher EE.
2.2. Particle Size (PS), Size Distribution, and External Morphology of F-LM
Based on an earlier study, a homogenization speed of 20,500 rpm was successfully used to fabricate
the F-LM particle size <10
µ
m (desirable size). Thus, a high speed homogenization (20,500 rpm) was
employed for further study.
From this study, Figure 3A shows the representative F-LM size distribution by laser particle
sixe (LPS) analyzer. The F-LM size distribution was a narrow curve corresponding to uniform sizes,
approximately 9
µ
m. This PS (9.18
µ
m) was achieved from the formulation of microsphere with
0.5% (w/v) PVA, 6.0% (w/v) PLGA, and 200
µ
g/mL fucoxanthin composition. From this study,
fabrication of F-LM by using 0.5% (w/v) PVA as surfactant, 6.0% (w/v) PLGA as a coating, and
200
µ
g/mL fucoxanthin as a core, produced discrete spheres with smooth surfaces and no pore
size (Figure 3B). Hong et al. [
24
] reported that the size distribution, PS, and pore size within the
microspheres are influenced by fabrication conditions such as the concentration of polymer, stirring
rate, good solvent/non solvent ratio, and the concentration of the dispersant.
Figure 3.
A representative of PS distribution of fucoxanthin-loaded microspheres (F-LM) by using
Laser Particle Size (LPS) analyzer (BT-9000H, Bettersize Instrument Ltd., China) (
A
), a representative of
external morphology of F-LM by using Field Emission Scanning Electron Microscope (FE-SEM) (JEOL,
JSM 6700F Model, Japan) with magnification 10,000×(B).
2.3. In Vitro Fucoxanthin Release Profile of F-LM
To investigate the effect of outer aqueous phase composition of F-LM on the
in vitro
fucoxanthin
release behavior of F-LM, a release test of F-LM in 0.1 M PBS (pH 7.4) at 37
C in static conditions was
performed.
In vitro
release was performed in Phosphate Buffered Saline (PBS) at pH 7.4 (bronchial
pH) and not at pH 5.2 (alveolar pH) [
23
] because the acidic pH can accelerate degradation of PLGA,
resulting in a reduced pH of the microenvironment [24].
Figure 4shows the
in vitro
fucoxanthin release profile of F-LM, and Figure 5show a standard
curve of fucoxanthin. The
in vitro
fucoxanthin release of F-LM was calculated based on this standard
curve. Figure 4represents the interaction between time intervals and cumulative (%) fucoxanthin
release. The curve of
in vitro
fucoxanthin release showed two profiles: A rapid release (burst release)
followed by a sustained release stage. Initially, a large burst release (38.3%) occurred at the first
hour. This effect may be associated with the presence of fucoxanthin crystals on or nearby the surface
of microspheres. A burst release was observed in this study because the polymer precursor did
not set immediately, causing unsuccessful encapsulation of some fucoxanthin, thus allowing free
fucoxanthin to release in a burst. The burst release is usually caused by fast desorption of the drug at
the surface [
17
]. Subsequently, high burst release is attributed to the microsphere porous structure,
which is commonly produced in the double w/o/wemulsion method [
25
27
]. In this case, the method
Molecules 2019,24, 947 8 of 16
used in this study was the w/o/wdouble emulsion extraction/evaporation method. However, the
burst release was significantly reduced by the immediate lyophilization of the harvested microspheres
following sonication-prepared emulsion [25].
Figure 4.
The curve of
in vitro
fucoxanthin release profile of F-LM as a function of time.
In vitro
fucoxanthin release was assessed by intermittently sampling the vial (1 mL) at predetermined time
intervals (1, 3, 6, 9, and 12 h, then 1, 2, 3, and 4 days, then 1, 2, and 3 weeks, then 1 and 2 months), and
was replaced with 1 mL of fresh 0.1 M PBS (pH 7.4) at 37 C.
Figure 5. Standard curve of commercial fucoxanthin (purity >95%).
Furthermore, in this study, the burst release step was followed by a gradual release of fucoxanthin,
reaching (79.1%) within 2 months. The F-LM had a high burst release (38.3%) due to PLGA (50/50)
used, and the small size of the microspheres. Tsai [
26
] reported that the microspheres with the lowest
molecular weight (MW) PLGA (50/50) showed the highest initial burst release compared to higher
MW of PLGAs (65/35; 75/25; or 85/15) microspheres [
28
] due to higher hydrophobic -CH
3
of lactic
acid (LA) in the composition.
The size ~9
µ
m of microspheres in this study affected the initial burst release of fucoxanthin.
Makadia and Siegel [
29
] reported that the size of PLGA microsphere also affected the initial burst
release. The drug release of smaller microspheres is faster than larger microspheres due to a higher
surface area-to-volume ratio [
13
,
30
]. Increased surface area enhances polymer and fucoxanthin
exposure to aqueous media, resulting in a larger initial burst and enhanced polymer degradation.
Moreover, the smaller microspheres have a shorter diffusion path length [
13
,
30
], thereby increasing
their penetration by the aqueous media [13,27].
Zhang and Zhu [
31
] have reported that, generally, microspheres prepared under various
conditions displayed similar release profiles, such as burst release, followed by a sustained release
stage. The initial burst release from microspheres might be due to the rapid release of the drug
Molecules 2019,24, 947 9 of 16
deposited on the microsphere surface [
32
]. This phenomenon occurs through the dissolution of the
drug, present at or near microsphere surfaces [
13
,
27
]. The initial burst of drug release is related to the
type of drug, the hydrophobicity of the polymer, and the concentration of drug [29].
Lewis [
33
] and Mao et al. [
34
] reported that the drug release pattern from PLGA microspheres
was biphasic, as a combination of the simple diffusion of the drug out of the polymer matrix and
erosion or degradation of the polymer matrix [
35
,
36
], which occurs via hydrolysis of the polymer back
bone [
37
]. Initially, the drug is released via diffusion through the polymer matrix, as well as through
the porous voids of the polymer structure [
38
]; but biodegradation of PLGA continuously changes
the drug release pattern [
32
]. The second process involves bulk erosion: The polymer matrix uptakes
water and polymer chains are degraded small enough to be soluble, and the drug is released during
the dissolution of the PLGA matrix [39].
It is well known that drug substances near the surface will diffuse out the microsphere first,
causing release [
32
]. The pattern of the result from this study was similar to the results obtained by
Emami [
40
], where insulin showed higher burst release (28%) and lower encapsulation efficiency (44%)
in PLGA microsphere prepared by w/o/wmethod.
2.4. Release Kinetics of F-LM
In order to determine the release model which best describes the pattern of fucoxanthin release
from F-LM, the
in vitro
fucoxanthin release data were substituted in zero order, first order, and Higuchi
model. Figure 6shows the zero order, first order, and Higuchi model of F-LM. Zero-order kinetics
describe a system where fucoxanthin release rate is independent of concentration and its cumulative
amount percentage of fucoxanthin release versus time (Figure 6A). The first-order kinetics describe the
release rate of fucoxanthin as dependent of concentration and its cumulative percentage of fucoxanthin
remains in the log scale versus time (Figure 6B). Higuchi model describes the release of fucoxanthin
from an insoluble matrix as a square root of time dependent process (Figure 6C).
Figure 6.
Zero-order kinetic data (
A
), first-order kinetic data (
B
), and Higuchi equation data
(C) of F-LM.
To confirm the pattern of fucoxanthin release,
in vitro
fucoxanthin release was subjected to release
kinetic studies based on its respective R
2
values, as given in Table 4. In this study, the
in vitro
release
kinetic of F-LM was best explained by the Higuchi equation, as the plots showed linearity (R
2
= 0.841),
Molecules 2019,24, 947 10 of 16
first order (R
2
= 0.792), followed by zero order equation (R
2
= 0.719). The best fit, with the highest
correlation in Higuchi equation, indicated that the release follows a diffusion-controlled pattern [
41
].
Thus, from this result, it is concluded that the release of fucoxanthin from PLGA (50/50) matrix was
predominantly controlled by Higuchi model.
2.5. Degradation Study of F-LM
Biodegradability analysis of F-LM was based on changes in surface morphology of the
microsphere. The changes in surface morphology of F-LM over time following incubation in 0.1 M PBS
(pH 7.4) at 37
C under static conditions is shown in Figure 7. An electron micrograph of F-LM before
incubation (control) showed a spherical, discrete microsphere with a smooth surface (Figure 7A).
Figure 7.
External morphologies change of F-LM dependent of time interval such as; control (
A
),
1 day (
B
), 1 week (
C
), 1 month (
D
), and 2 months (
E
) by using FE-SEM (JEOL, JSM 6700F Model) with
magnification 1500×.
As illustrated in Figure 7B, minor changes in F-LM morphology were observed during the first
hour. F-LM showed progressive pores emerging on the surface that describe the burst release phase.
Following this, major changes in F-LM morphology were observed within 1 week and the F-LM had
deformed, aggregated, and fused (Figure 7C), and by 1 month (Figure 7D), the F-LM morphologies
had turned into an unstructured mass. Finally, the F-LM were totally collapsed and disintegrated
into irregular particles, with no intact spheres observed (Figure 7E). The F-LM fabricated from PLGA
(50/50) as coating, needed 2 months for degradation. Lewis [
33
] reported that controlled release of
a desired drug is over a period of 1 to 3 months. Vidyavathy and Ramana [
42
] and Middleton and
Tipton [
43
] reported that PLGA (50/50) polymer degraded in approximately 1 to 2 months, and PLGA
75/25 and PLGA 85/15 degraded in 4 to 5 months and 5 to 6 months, respectively.
PLGA polymer can be degraded into oligomeric and finally monomeric acids [
42
44
], such
as lactic acid and glycolic acid that are non-toxic to the human body [
45
], and can be completely
biodegraded into CO
2
and H
2
O [
46
]. Furthermore, in the human body, polyglycolides are broken
down into glycine which can be excreted in the urine or converted into CO
2
and H
2
O via the citric
acid cycle [47,48].
Molecules 2019,24, 947 11 of 16
3. Materials and Methods
3.1. Materials
All chemicals used in this study were of analytical grade. Poly(D,L-lactic-co-glycolic acid) (PLGA)
(Purasorb PDLG 5004) was purchased from PURAC (Gorinchem, The Netherlands), polyvinylalcohol
(PVA) with a molecular weight (MW) of 115 kDa was purchased from BDH laboratory supplies (Poole,
UK), Tween 80 was purchased from MERCK (Darmstadt, Germany), phosphate buffer saline (PBS),
fucoxanthin (purity >95%) were supplied by Sigma-Aldrich, dichloromethane (DCM) was purchased
from Fisher Scientific (Leicestershire, UK).
3.2. Optimization of Medium Component by RSM
RSM was used to optimize the PS and EE of the w/o/wdouble emulsion evaporation method.
A FCCCD developed by Design Expert software (version 6.0.8, Stat-Ease Inc., Minneapolis, MN,
USA) [
49
] was used to optimize three significant extraction conditions: PVA, PLGA, and fucoxanthin
concentration for optimum PS and EE. A set of 15 experimental runs with one center point (run 12) was
generated. Subsequently, three different levels, low (
1), medium (0), and high (+1) were used to study
the independent variables. The PS and EE were considered as the response (Y
1
) and (Y
2
), respectively.
The following second-order polynomial equation explains the relationship between dependent and
independent variables [49]:
Y1or Y2=β0+β1A+β2B+β3C+β11A2+β22 B2+β33C2+β12AB +β13 AC +β23BC (3)
where Y
1
is the dependent variable (particle size, PS), and Y
2
is the dependent variable (encapsulation
efficiency, EE); A,B, and Care independent variables PVA, PLGA, and fucoxanthin concentration,
respectively;
β0
is an intercept term;
β1
,
β2
, and
β3
are linear coefficients;
β12
,
β13
, and
β23
are the
interaction coefficients; and β11,β22, and β33 are the quadratic coefficients.
The developed regression model was evaluated by analyzing the values of regression coefficients,
analysis of variance (ANOVA), p- and F-values (www.waset.org). The quality of fit of the polynomial
model equation was expressed by the coefficient of determination, R
2
[
50
,
51
]. Furthermore, to explain
the relationship between the experimental levels of each of variables under study and the responses,
the fitted polynomial equation was expressed in the form of 3D response surface and 2D contour [
49
].
3.3. Fabrication of F-LM
A double-emulsion solvent evaporation method was adopted from Mohamed and Walle [
52
] with
some modifications. Briefly, 178
µ
L of dH
2
O was mixed with 22
µ
L of PVA, producing 0.5% w/vof
aqueous phase. This aqueous phase was added into both 120 mg of PLGA (50/50) and 400
µ
g/mL
of fucoxanthin previously dissolved in 2 mL DCM (oil phase). This mixture was homogenized at
20,500 rpm (IKA
®
T10 basic Ultra-Turrax, Kuala Lumpur, Malaysia) for 3 min (primary emulsion, PE).
After homogenization, PE was immediately subjected to 22 mL 0.5 (% w/v) PVA of 10 times the volume
of PE [
53
]. Following this, the mixture was homogenized again at 20,500 rpm for 10 min to produce the
secondary emulsion (SE). Subsequently, this SE was transferred into a continuously stirred hardening
tank [
53
] containing 100 mL of 0.5% PVA. This stirring was continued for 2 to 3 h [
54
] to allow complete
evaporation of DCM. The hardened microspheres were collected by centrifugation [
26
] (2500 rpm),
washed with 600 mL distillated water and then lyophilized overnight [
53
]. Lyophilized microspheres
were kept at
20
C in an air-tight container with silica gel until further evaluation [
53
55
]. The
general microsphere formulation recipe is shown in Table 1.
3.4. EE of F-LM
An accurate amount of lyophilized F-LM (2 mg) was suspended in 1 mL PBS to which 1 mL
acetone was added to solubilize PLGA. The tube was centrifuged at 10,000 rpm for 3 min. The
Molecules 2019,24, 947 12 of 16
supernatant (100
µ
L) was transferred to CELLSTAR
®
96 well plate flat bottom (Greiner bio-one, Kuala
Lumpur, Malaysia) [
53
] and analyzed using a microplate reader (Tekan/Infinite M200, NanoQuant,
Kuala Lumpur, Malaysia) by measuring absorbance at 450 nm (maximum wave length for detecting
fucoxanthin) [
56
]. The absorbance values were substituted into a standard curve of linear regression of
known free fucoxanthin concentrations to obtain the actual concentrations of extracted fucoxanthin
from F-LM. EE was calculated based on the ratio of the actual fucoxanthin concentration to theoretical
loading, expressed as percentage [53].
3.5. Particle Size Analysis of F-LM
An LSP analyzer (BT-9300H, Better Size Instrument Ltd., Shanghai, China), which is a laser
diffractometer was used to determine the size distribution of the microspheres prior to lyophilization,
dispersing the microspheres in water until approximately 25% obscurity was reached. The size
distribution was expressed as volume median diameter (VMD) [
52
]. Data are presented as d(0.5)
which is equivalent volume diameter at 50% cumulative volume.
3.6. External Morphology of F-LM
A FE-SEM (JEOL, JSM 6700F Model) was used to capture images for evaluation of shape, size,
and external morphology of the F-LM. Briefly, a small amount of lyophilized F-LM was mounted on
aluminum stubs pre-pasted with double-sided copper tapes. The sample was sputter-coated with a
thin layer of gold and placed inside the specimen chamber at an accelerating voltage of 3 kV at 20
C
and 105Torr [53].
3.7. In Vitro Fucoxanthin Release Profile
Lyophilized F-LM (2.5 mg) was accurately weighed and added into vials containing 1 mL PBS
(pH 7.4) with 0.01 % (w/v) Tween-80 to improve solubility of the drug [
57
]. The vials were kept at
37
C without agitation.
In vitro
fucoxanthin release was assessed by intermittently sampling the vials
(1 mL) at predetermined time intervals [
57
] (1, 3, 6, 9, and 12 h, then 1, 2, 3, and 4 days, then 1, 2, and
3 weeks, then 1 and 2 months), and was replaced with 1 mL of fresh pH 7.4 phosphate buffer [
22
]. The
withdrawn sample was centrifuged at 10,000 rpm for 3 min. The supernatant was then collected and
transferred to CELLSTAR
®
96 well flat bottom plate (Greiner Bio-one, Kuala Lumpur, Malaysia) [
53
]
and read by a microplate reader (Tekan/Infinite M200, NanoQuant, Kuala Lumpur, Malaysia) with
visible absorbance measurement at 450 nm (maximum wave length for detecting fucoxanthin) [
56
].
The amount of fucoxanthin released in each sample was determined using a curve of calibration; the
reported values are averages of three replicates (n = 3). Results of
in vitro
fucoxanthin release studies
obtained were tabulated and shown graphically as cumulative % drug release versus time [57].
3.8. Evaluation of Release Kinetics
The mechanism and kinetics of fucoxanthin release from F-LM was analyzed using mathematical
models [58] such as zero-order kinetics, first-order kinetics and Higuchi kinetics (Table 5).
Table 5. Mathematical equations for the models used to describe release kinetic of drugs.
Model Plot Equation
Zero order Qtvs. t Qt= K0t
First order ln (Q0Qt) vs. t ln Qt= ln Q0K1t
Higuchi Qtvs. t1/2 Qt= Kht1/2
3.9. Degradation Study of F-LM
Degradation study method was adopted from Wang [
59
] with some modifications. Briefly,
pre-weighed F-LMs (about 2.5 mg) were placed in individual vial tubes (15 vials) containing 1.0 mL of
Molecules 2019,24, 947 13 of 16
PBS (pH 7.4). The vial tubes were kept in an incubator that was maintained at 37
C. At predetermined
degradation intervals (0 day as control, 1 day, 1 week, 1 month, and 2 months, respectively) the F-LMs
were collected by centrifugation, washed with distilled water to remove residual buffer salt, and
freeze-dried overnight. Following this, the surface morphology of degraded F-LM was analyzed using
FE-SEM (JEOL, JSM 6700F Model, Tokyo, Japan) at 3.0 kV [60].
4. Conclusions
The physical and chemical nature of fucoxanthin affects its potency and effective delivery as
an anti-cancer drug against H1299 human lung cancer cells. Microencapsulation is an attractive
drug delivery method to overcome this problem. In this study, fucoxanthin was successfully
microencapsulated using double emulsion extraction/evaporation method to produce F-LM. The use
of RSM optimized the PS and EE of the F-LM. F-LM with the best response was fabricated using a
formulation of 0.5% (w/v) PVA, 6.0% (w/v) PLGA and 200
µ
g/mL fucoxanthin with a homogenization
speed of 20,500 rpm. Under these conditions, F-LM with PS (9.18
µ
m) and EE (33.09%) was produced.
The three factors chosen in RSM have shown significant effects on the response in PS and EE. The PS
and EE in turn, affect the drug release and degradation characteristics of the F-LM. The optimization
of process parameters is an important consideration in the production of drug microspheres. PS,
distribution, and morphology, as well as an understanding of the kinetics and degradation pattern of
the microspheres, translate into more precise control of the drug release. The information obtained
from this study is important for improving efficacy and potential for commercialization of fucoxanthin
as an anticancer drug in the near future.
Author Contributions:
Conceptualization, I.J., D.N., M.H., F.O., W.L.; Methodology, I.J., D.N., M.H., F.O.; Software,
I.J.; Validation, I.J., D.N., M.H., A.G.M.; Formal analysis, I.J., M.H., F.H., F.O.; Investigation, I.J., D.N., M.H., F.O.,
S.N., B.B.H.A.; Resources, I.J., F.O., A.G.M., S.N., B.B.H.A.; Data curation, I.J., D.N., B.B.H.A.; Writing—original
draft preparation, I.J., D.N.; Writing—review and editing, I.J., F.O., W.L., A.G.M., S.N., B.B.H.; Visualization, I.J.,
D.N.; Supervision, I.J., A.G.M., S.N., B.B.H.A. W.L.; Project administration, I.J.; Funding acquisition, I.J.
Funding:
This research was funded by the Ministry of Higher Education Malaysia, KIHIM Research Grant
MOHE 18-002-0002.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds fucoxanthin are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... Of particular interest are carotenoids of marine origin, since their biological activity makes it possible to create highly effective drugs for the prevention and treatment of severe diseases. For example, fucoxanthin (Fx), the share of which in C. closterium biomass reaches 2% of dry weight, is a unique carotenoid with an allene bond that exhibits high antitumor activity [3,4], as well as cytotoxic activity against prostate cancer cells PC-3, DU145 and LNCap [5,6]; induces apoptosis of leukemic cells HL-60 and colon cancer cells Caco-2, HT-29, and DLD-1 [5][6][7]; stops the cell cycle in GOTO neuroblastoma cells; etc. ...
... Of particular interest are carotenoids of marine origin, since their biological activity makes it possible to create highly effective drugs for the prevention and treatment of severe diseases. For example, fucoxanthin (Fx), the share of which in C. closterium biomass reaches 2% of dry weight, is a unique carotenoid with an allene bond that exhibits high antitumor activity [3,4], as well as cytotoxic activity against prostate cancer cells PC-3, DU145 and LNCap [5,6]; induces apoptosis of leukemic cells HL-60 and colon cancer cells Caco-2, HT-29, and DLD-1 [5][6][7]; stops the cell cycle in GOTO neuroblastoma cells; etc. ...
... It was centrifuged (3 min, 8000 rpm) and the supernatant was diluted for testing. The EE of FX was evaluated as follows [46]: ...
... The measurement method was the same as that for total FX determination in 3.5. LC was calculated using the following formula [46]: ...
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Fucoxanthin (FX) is a marine carotenoid that has proven to be a promising marine drug due to the multiple bioactivities it possesses. However, the instability and poor bioavailability of FX greatly limit its application in pharmaceuticals or functional foods. In this study, the creative construction of a solid lipid nanoparticle-microcapsule delivery system using mixed lipids of palm stearin and cholesterol wrapped with gelatin/Arabic gum to load lipophilic FX was fabricated, aiming to improve the stability and bioavailability of FX. The results showed that the encapsulated efficiency (EE) and drug loading capacity (LC) of optimized FX microcapsules (FX-MCs) obtained were as high as 96.24 ± 4.60% and 0.85 ± 0.04%, respectively, after single-factor experiments. The average particle size was 1154 ± 54 nm with negative Zeta potential (−20.71 ± 0.93 mV) as depicted with size-zeta potential spectrometer. The differential scanning calorimeter (DSC) and thermogravimetric analyzer (TG) results indicated that FX-MC has a higher Tg and slower weight loss than FX monomers (FX crystal) and blank MCs. Besides, The Fourier transform infrared spectrometer (FTIR) confirmed the good double encapsulation of FX into the solid lipid and composite coacervate. Moreover, the encapsulated FX showed higher storage stability, sustained release (55.02 ± 2.80% release in 8 h), and significantly improved bioavailability (712.33%) when compared to free FX. The research results can provide a principle theoretical basis for the development and application of FX in pharmaceuticals or functional foods.
... A face-centred (α = 1) three-level, two-factor central composite design with a quadratic model was developed using Design Expert Software ® (version 8.0.3.1 Stat-Ease Inc.). The minimum number of trials needed to optimize the microparticle formulation was determined using this model [22,23]. Considering earlier study and the literature survey [24,25] the concentration of sodium alginate (X 1 ) and the stirring speed (X 2 ) were considered as independent formulation (or process) parameters. ...
... Formulation B2 was the optimized formulation. The experimental values of both the independent and dependent variables of formulation B2 are in accordance with the software-generated theoretical values [21,22,25]. Response surface plots were drawn to study the effects of the independent variables on the resultant responses, namely the % production yield, %EE and % mucoadhesion strength. ...
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Background Oxybutynin chloride (OXC) is used to treat overactive urinary bladder. OXC is metabolized in the liver to N-desethyloxybutynin, which is mainly responsible for the anticholinergic side effects of OXC. Conventional oxybutynin suppositories formulated earlier have shown most common side effects, such as dry mouth, constipation and serious anticholinergic reaction. Hence the present research work deals with the formulation and characterization of OXC microparticle-loaded mucoadhesive suppositories which may remain adhered in the lower rectum and avoid first pass metabolism. The emulsification–ionic gelation method is employed to prepare OXC microparticles. Two formulation factors at three levels, i.e. polymer concentration and stirring speed, were selected. Sodium alginate (concentration 1–2%) and 1% w/v Carbopol 971P were used to prepare OXC microparticles. OXC microparticles were evaluated for various parameters such as production yield, entrapment efficiency, mucoadhesive strength, shape, size, zeta potential, Fourier Transform Infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, in vitro dissolution studies and stability studies. Suppositories loaded with OXC microparticles were prepared by the fusion method using Poloxamer 188 and propylene glycol and evaluated for various parameters like weight variation, disintegration time, in vitro dissolution study, stability study and pharmacokinetic study. Results Results of in vitro characterization revealed that optimized batch of OXC loaded microparticles exhibited production yield 94.024% entrapment efficiency 95.378% and mucoadhesion strength 95.544%, particle size range 764.04–894.13 µm, zeta potential − 14.5 mV, with 0.946 desirability. Consequences of DSC and XRPD evaluation shown that drug was effectively entrapped inside the microparticles. In vitro release studies revealed improvement in drug dissolution as a consequence of its entrapment into microparticles. SEM results showed that micelles were sphere-shaped. On rectal administration of OXC microparticles loaded suppository in male Sprague–Dawley Rats, the relative bioavailability was found 173.72%. Conclusion In vivo study elicits rapid increase in absorption of drug from microparticles loaded suppository when compared with the oral formulation and drug loaded suppository in rats. OXC microparticles loaded suppository is novel and promising drug delivery system for rectal administration and may avoid anticholinergic side effects of hepatic metabolite, N-desethyloxybutynin. These rectal drug delivery systems will be advantageous for efficient absorption of drugs and to avoid first pass metabolism.
... We note that the final concentration of fucoxanthin achieved after 7 extraction cycles is substantially higher than the loading of 0.1-0.3 mg/mL proposed for therapeutic applications of fucoxanthin [47]. Given the DES components are safe food additives, this highlights the potential to use these extracts as concentrates that could be diluted to appropriate concentrations for therapeutic use. ...
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Fucoxanthin is a carotenoid in algae with purported beneficial health-related properties including antioxidant, anti-photoaging, anti-metastasis, anti-hypertensive activity and more. These properties give fucoxanthin the potential to be used in cosmetic, dietary, and medicinal applications. This study evaluates the use of deep eutectic solvents (DESs) to extract fucoxanthin from the microalgae Tisochrysis lutea. Conductor-like Screening Model for Real Solvents (COSMO-RS) was used to screen the performance of 24 different types of DESs in the extraction of fucoxanthin based on their calculated capacities. Experimental extraction validation was then carried out using the 6 top-ranked DESs. The experimental results revealed that the extraction capacity of the thymol: dodecanoic acid DES (1.25: 1 molar ratio) for fucoxanthin was the most efficient (7.69 mg/g dry biomass weight (DW)) among the DESs explored under the screening conditions and was higher than the capacity of the conventional solvents methanol (6.29 mg/g DW) and ethanol (6.75 mg/g DW). This corresponded with COSMO-RS screening results. Then, the optimisation of extraction conditions for fucoxanthin using thymol: dodecanoic acid DES was further investigated, revealing that the highest yield of fucoxanthin (22.03 mg/g DW) was extracted at optimum experimental conditions at a temperature of 36.2 ℃, stirring time of 2.58 h, and the biomass percentage of 11.36%. Additionally, fucoxanthin showed good stability in thymol: dodecanoic acid DES over eleven days of storage. After seven extraction cycles, the final fucoxanthin concentration (13.06 mg/mL DES) resulted in a good reusability of the terpene-based food safe DES.
... Nevertheless, R-PE was denatured and exhibited dual-color fluorescence emission including green and red light, while the original single orange light was significantly suppressed [24]. Finally, also FX stability was assessed under encapsulation in polyvinylpyrrolidone nanoparticles [25] or PVA and poly(D,L-lactic-co-glycolic acid) microspheres [26]. In line with the recent interest in biomolecules for solar-based applications, namely solar harvesting sensors [5] and LSCs [6], the interest in new biomolecules with enhanced solar absorption and conversion and stability gained visibility. ...
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In the search for a better and brighter future, the use of natural luminescent renewable materials as substitutes for synthetic ones in the energy field is of prime importance. The incorporation of natural pigments (e.g., xanthophylls and phycobiliproteins) is a fundamental step in a broad spectrum of applications that are presently marred by their limited stability. The incorporation of bio-based luminescent molecules into solid matrices allows the fabrication of thin films, which may dramatically increase the range of applications, including sustainable photovoltaic systems, such as luminescent solar concentrators or downshifting layers. In this work, we incorporated R-phycoerythrin (R-PE), C-phycocyanin (C-PC), and fucoxanthin (FX) into poly(vinyl alcohol) (PVA) and studied their optical properties. It was found that the emission and excitation spectra of the phycobiliproteins and FX were not modified by incorporation into the PVA matrix. Moreover, in the case of FX, the emission quantum yield (η) values also remained unaltered after incorporation, showing the suitability of the PVA as a host matrix. A preliminary photostability study was performed by exposing the solid samples to continuous AM1.5G solar radiation, which evidenced the potential of these materials for future photovoltaics.
... Y = b o + b 1 × 1 + b 2 X 2 + b 11 X 1 2 + b 22 X 2 2 + b 12 X 1 X 2 where Y = response; X 1 = PAG concentration; X 2 = Alginate concentration; b o = intercept; b 1 -b 5 = co-efficients; X 1 2 /X 2 2 = second order effects of PAG and ALG concentrations respectively; X 1 X 2 = interactive effects of PAG and ALG concentrations. The optimized formulations were selected based on the desirability parameter and validated for the calculated statistical model by ANOVA (analysis of variance) before subjecting all to further studies [43,44]. ...
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Combinations of polymers can improve the functional properties of microspheres to achieve desired therapeutic goals. Hence, the present study aimed to formulate Prunus armeniaca gum (PAG) and sodium alginate microsphere for sustained drug release. Blended and coated microspheres were prepared using the ionotropic gelation technique. The effect of polymer concentration variation was studied on the structural and functional properties of formulated microspheres. FTIR, XRD, and thermal analysis were performed to characterize the microspheres. All the formulations were well-formed spherical beads having an average diameter from 579.23 ± 07.09 to 657.67 ± 08.74 μm. Microspheres entrapped drugs within the range 65.86 ± 0.26–83.74 ± 0.79%. The pH-dependent swelling index of coated formulations was higher than blended. FTIR spectra confirmed the presence of characteristic peaks of entrapped Tramadol hydrochloride showing no drug-polymer interaction. In vitro drug release profile showed sustained release following the Korsmeyer-Peppas kinetic model with an R2 value of 0.9803–0.9966. An acute toxicology study employing the oral route in Swiss albino mice showed no signs of toxicity. It can be inferred from these results that blending PAG with sodium alginate can enhance the stability of alginate microspheres and improve its drug release profile by prolonging the release time.
... From the results, linear polynomial equation was used to determine the dependence of responses on factors, shown in Table 1a. The positive values (ALG and QSM) indicating an improvement in responses (DEE, mean diameter and % yield) whilst the negative values indicated that response efficiency doesn't increase with factor improvement (Afzalinia & Mirzaee, 2020;Jaswir et al., 2019). Additionally, there was a good agreement between predictable and actual values which advocated the adequacy of models, Fig. 2. The linear model was found to be adequate for DEE % and % yield whereas 2FI was approved for mean diameter, equations shown in Table 1a and 1b. ...
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Quince seed mucilage was used in a combination of sodium alginate to develop sustained-release microspheres of cefixime. Physical characterizations such as FTIR, TGA, DSC, and SEM were performed on the prepared microspheres. The swelling of microspheres was maximum at pH 7.4 and reduced at acidic pH. The average particle size ranged from 679 µm ±0.21 to 810 µm ±0.31, while the drug encapsulation efficiency range was found as 73.76±0.24 to 85.63±0.46. In vitro release profile of QSM-alginate-cefixime microspheres followed Korsmeyer-Peppas model (R² = 0.9732-0.9946); and release was non-Fickian as we found value of n > 1. This study reveals the benefits of QSM-alginate microspheres for the sustained release of cefixime without any toxicity and it also improved antibacterial properties.
... A negative value in quadratic equation indicates an inverse relationship or antagonistic effect between the factor and the response while a positive value in the regression equation exhibits an effect that favors the optimization due to synergistic effect. A negative sign in quadratic formula implies a negative correlation or antagonistic action whilst the positive value in regression model demonstrates synergistic effect of factors on response variables [31,32] . This might be linked to the fact that PVP K-30 and Poloxamer 407 have solubilizing effect on hydrophobic drug particles due to their characteristics of decreasing surface tension and increasing wettability and dispersibility. ...
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Mangosteen fruit has been widely consumed and used as a source of antioxidants, either in the form of fresh fruit or processed products. However, mangosteen peel only becomes industrial waste due to its bitter taste, low content solubility, and poor stability. Therefore, this study aimed to design mangosteen peel extract microcapsules (MPEMs) and tablets to overcome the challenges. The fluidized bed spray-drying method was used to develop MPEM, with hydroxypropyl methylcellulose (HPMC) as the core mixture and polyvinyl alcohol (PVA) as the coating agent. The obtained MPEM was spherical with a hollow surface and had a size of 411.2 µm. The flow rate and compressibility of MPEM increased significantly after granulation. A formula containing 5% w/w polyvinyl pyrrolidone K30 (PVP K30) as a binder had the best tablet characteristics, with a hardness of 87.8 ± 1.398 N, friability of 0.94%, and disintegration time of 25.75 ± 0.676 min. Microencapsulation of mangosteen peel extract maintains the stability of its compound (total phenolic and α-mangosteen) and its antioxidant activity (IC50) during the manufacturing process and a month of storage at IVB zone conditions. According to the findings, the microencapsulation is an effective technique for improving the solubility and antioxidant stability of mangosteen peel extract during manufacture and storage.
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Purpose of writing this review on microspheres was to compile the recent literature with special focus on formulation variables which affect the drug entrapment efficiency of microspheres. There are various approaches in delivering a therapeutic substance to the target site in a controlled release fashion. One such approach is using microspheres as carriers for drugs. Microencapsulation is used to modify and delayed drug release form pharmaceutical dosage forms. For success of microspheres as drug delivery system its necessary to obtained desired particle size, maximum drug entrapment, mucoadhesion, swelling index and drug release. This can be obtained by optimizing the formulation as well as process variables but before designing the microspheres formulation deep understanding the effect of various variables on characteristics of microspheres is necessary. The intent of the paper is to highlight the reported study on various formulation variables those are might be useful to encountered several problems which is reason for low drug entrapment efficiency
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The aim of the present study is to develop a multiparticulate system containing pectin microspheres for the colon targeted delivery of Tramdol HCl (TMD) for the treatment of irritable bowel syndrome. This work combines pH-dependent solubility of shellac polymers and microbial degradability of pectin polymers. Pectin microspheres containing TMD were prepared by emulsion cross linking method using different ratios of TMD and pectin (1:2 to 1:5), stirring speeds (500-2000 rpm) and emulsifier concentrations (1.0% - 2.0% wt/vol). The yield of preparation and the encapsulation efficiencies were high for all pectin microspheres. Microspheres prepared by using drug: polymer ratio 1:3, stirring speed 1000 rpm, and 1.25% wt/vol concentration of emulsifying agent were selected as an optimized formulation. Shellaccoating of pectin microspheres was performed by oil-in-oil solvent evaporation method using coat: core ratio (5:1). Microspheres were evaluated for surface morphology, particle size and size distribution, swellability, percentage drug entrapment, and in vitro drug release in simulated gastrointestinal fluids (SGF). The release profile of TMD from Shellac-coated pectin microspheres was pH dependent. In acidic medium, the release rate was much slower; however, the drug was released quickly at pH 7.4. It is concluded from the present investigation that Shellac-coated pectin microspheres are promising controlled release carriers for colon-targeted delivery of TMD.
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In this study, the effects of stirring rates (8000, 9500, 13500 rpm) and drug:Polymer ratios (1:25, 4:25, 6:25) on the pharmaceutical characteristics of Levobunolol HCl-loaded poly (s-caprolactone) microparticles in order to optimize formulations for ocular delivery were examined. Microparticles were prepared using multiple emulsion (W/O/W) solvent evaporation technique. Microparticles were characterized in terms of surface morphology, drug loading efficiency, production yield, particle size and size distribution, drug release, zeta potential and residual solvent. The physical state of microparticles was also determined by scanning electron microscopy (SEM). Spherical, smoother particles were obtained with a drug:polymer ratio of 6:25 at 8000 rpm. Drug:polymer ratio played greater role on drug loading efficiency than stirring rate. An increase in the stirring rate from 8000 to 9500 rpm resulted a decrease in particle size at all drug:polymer ratios. The fasted drug release and the highest drug-loading efficiency obtained from formulation of A10 prepared with 1:25 drug:polymer ratio at the stirring rate of 8000 rpm. Drug release was mainly fitted to the Higuchi model. The findings showed that both stirring rates and drug:polymer ratios had an effect on the pharmaceutical properties of microparticles.
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Colorectal cancer therapy with 5-fluorouracil (5-Fu) frequently become ineffective due to resistance to this drug; and thus other effective compounds are essential for therapy. It is well-known marine brown seaweeds contain antioxidant compounds the carotenoid fucoxanthin (Fx) and polyphenolic compound phloroglucinol (Ph) which exerted diverse biological activities including antioxidant and anticancer. The aim of this study was to determine the anticancer activities of Fx or Ph alone as well as combination of each chemical with 5-Fu on two human colorectal cancer cell lines (HCT116 and HT29), with comparison to responses in a normal colon cell line (CCD-18Co). Effects of these compounds on cell viability, induction of DNA damage, and cell death were evaluated using MTT assay, comet assay, nuclear condensation assay, and Western blot. 5-Fu decreased cell viability in a concentration-dependent manner in HCT116 and HT29 cells but was not cytotoxic in CCD-18Co cells. 5-Fu induced DNA damage in HCT116 cells with induction of cell death, while no marked effects on DNA damage and cell death were observed in HT29 cells. Fx or Ph alone also reduced cell viability in both cancer cell lines but no apparent cytotoxic effect in CCD-18Co cells, except for Fx at 50 and 100 µM. Diminished cell viability was accompanied by induction of DNA damage (by Fx) and induction of cell death (by Ph). In combination with 5-Fu, Fx at 10 µM (in HCT116 and HT29 cells), and Ph at 300 µM (in HT29 cells) enhanced the cytotoxic effect of 5-Fu; however, no marked cytotoxicity was noted in CCD-18Co cells. Since Fx and Ph alone reduced cancer cell line viability without an effect on normal cells and when in combination enhanced the cytotoxic effect of 5-Fu only in colon cancer cells, these compounds seem promising as anticancer agents.
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Fucoxanthin is a marine carotenoid mainly found in brown seaweeds. Its antitumor and cancer-preventative function has been extensively investigated. Investigations have indicated that fucoxanthin and its metabolite fucoxanthinol induce G1 cell-cycle arrest and apoptosis in various cell lines and can inhibit cancer development in animal models. It is imperative that the underlying mechanism of action of fucoxanthin be elucidated in order to facilitate the development of cancer-prevention strategies in humans. Key molecules that require consideration include mitogen-activated protein kinase, growth arrest and DNA damage-inducible 45, AP-1 transcription factor, nuclear factor-kappa B and several others, including cell cyclerelated molecules for G1 cell-cycle arrest and the B cell lymphoma-2 family, X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein and AKT serine/threonine kinase/phosphatidylinositol-3-kinase for apoptosis. In this review, the mechanisms by which fucoxanthin exerts its antitumor and cancer-preventative action in cell lines and mouse models is discussed, in addition to the potential use of fucoxanthin as a promising compound for cancer prevention.
Thesis
Investigation on targeted PLGA based drug delivery system for the therapy of colorectal cancer. The results from in-vitro cell experiments indicated that prepared systems have potent cytotoxicity and high affinity to HT-29 cancer cells. Results were published on Biomedical Engineering and Informatics and ICONN conference proceeding. <br /
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N-Palmitoylethanolamide showed great therapeutic potential in the treatment of inflammation and pain but its unfavourable pharmacokinetics properties will hinder its use in the clinical practice. A nanotechnology-based formulation was developed to enhance the probability of N-palmitoylethanolamide therapeutic success, especially in skin disease management. Lipid nanoparticles were produced and characterized to evaluate their mean size, ζ-potential, thermal behaviour, and morphology. The ability of N-palmitoylethanolamide to diffuse across the epidermis as well as anti-inflammatory and analgesic effects were investigated. Particles had a mean size of about 150nm and a ζ-potential of -40mV. DSC data confirmed the solid state of the matrix and the embedding of N-palmitoylethanolamide while electron microscopy have evidenced a peculiar internal structure (i.e., low-electrondense spherical objects within the matrix) that can be reliably ascribed to the presence of oil nanocompartments. Lipid nanoparticles increased N-palmitoylethanolamide percutaneous diffusion and prolonged the anti-inflammatory and analgesic effects in vivo. Lipid nanoparticles seem a good nanotechnology-based strategy to bring N-palmitoylethanolamide to clinics.
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The aim of the present study was to prepare matrix pellets loaded nifedipine (NF) as model drug by pellitization technique by using blend of gelucire 50/13 (GL) and glyceryl palmito stearate (GPS) as hydrophilic and hydrophobic carriers in different concentrations. This system was able to prolong the drug release, minimizing the drug related adverse effects and improve bioavailability in different GI-tract conditions. The prepared formulations was subjected to micromeritic properties, SEM, DSC, FTIR and stability studies. The obtained microspheres having smooth surfaces, with free flowing and good packing properties, angle of repose, % Carr's index and tapped density values were within the limit. The drug loaded in microspheres was stable and compatible, as confirmed by DSC and FTIR studies. In-vitro drug release profile of NF from pellets was studied in simulated gastric fluid pH1.2 for initial 2h and in simulated gastric fluid pH 7.4 upto 22 h and compared with oral formulation Adalat CR® 20 capsule. The release of drug from the pellets showed negligible drug release in pH1.2. Under intestinal conditions resulting optimum level of drug released in a controlled manner and exhibited fickian diffusion.