Pinheiro, A. C.

Vicente, A. A.

Universidade do Minho: RepositoriUM

Semana de Engenharia 2010 
Guimarães, 11 a 15 de Outubro 
 
CONTROLLED RELEASE OF A MODEL BIOACTIVE COMPOUND FROM -
CARRAGEENAN/CHITOSAN NANOLAYERED FILMS 
 
Ana C. Pinheiro, António A. Vicente 
Department of Biological Engineering 
E-mail: anapinheiro@deb.uminho.pt 
 
 
KEYWORDS 
 
Multilayers, layer-by-layer, transport modeling  
 
ABSTRACT 
 
Nanotechnology holds a great potential to 
generate very innovative solutions for the food 
industry and packaging is one of the many areas 
that can benefit from this new technology. The 
main goal of this work is to prepare nanofilms 
by layer-by-layer deposition using κ-
carrageenan and chitosan, incorporate the model 
compound methylene blue and to evaluate its 
loading and release behaviour. The developed 
biodegradable nanofilms are a promising 
delivery system for application in food products, 
as a strategy for shelf-life extension. 
 
INTRODUCTION 
 
Layer-by-layer (LbL) assembly is based on the 
alternating deposition of oppositely charged 
polyelectrolytes and can be used to produce 
multilayers of nanometer thickness. 
Nanolayered films have promising applications 
on food products, as a strategy for shelf-life 
extension. However, there is a need to create 
multilayer systems that combine both 
biofuncionality and edibility. Chitosan, a natural 
cationic polysaccharide, is an excellent edible 
film component due to its good oxygen barrier 
properties and due to its intrinsic antimicrobial 
activity (Bégin & Van Calsteren, 1999). K-
carrageenan, an anionic polysaccharide, is 
extensively used in food industry as gelling and 
stabilizing agent and has been reported as 
having excellent film forming properties (Park 
et al., 1996). In recent years, LbL nanolayered 
films have been investigated due to the 
possibility of controlling drug release (Wang et 
al., 2007). Furthermore, drug molecules can act 
as either functional drugs or components of the 
film. The release behaviour of bioactive 
compounds from nanolayered films depends on 
the permeability, on the disassembly or erosion 
of the multilayer structure and on other 
experimental variables (e.g. pH) (Jiang & Li, 
2009). Methylene blue (MB), a monovalent 
cation of 374 Da, is a suitable model for loading 
and release experiments due to its large 
absorption peak in the visible range. MB has 
been used to investigate multilayer surface 
properties due to its ability to bind to available 
negatively charged functional groups (Chung & 
Rubner, 2002).  
In this work multilayer nanofilms were prepared 
by LbL deposition using κ-carrageenan and 
chitosan onto a substrate (polyethylene 
terephthalate - PET). MB was incorporated in 
the nanolayered film and its loading and release 
behaviour was evaluated. 
 
METHODS 
 
Preparation of nanolayered film: The κ-
carrageenan and the chitosan solutions were 
prepared at a concentration of 0.2 % (w/v) in 
distilled water and 1 % acid lactic, respectively. 
The solutions’ pH was adjusted to 7 and 3, 
respectively. MB solution was prepared at a 
concentration of 0.3 % (w/v) in 50 % (v/v) PBS 
and the pH was adjusted to 7. The nanolayered 
film was composed by an aminolyzed PET 
sustaining layer adsorbed with 5 layers (3 κ-
carrageenan and 2 chitosan layers). The MB 
was incorporated in the film, forming the 
second, the fourth or the sixth layer, thus 
replacing the corresponding chitosan layer in 
those positions. The formation of nanolayered 
film was performed according to Carneiro-da-
Cunha et al. (2010). 
MB loading: The loading of MB on each 
position of the multilayer structure was 
monitored by UV-VIS spectroscopy (Jasco 560, 
Germany). Real time deposition of MB on the 
coated electrode was recorded by a Quartz 
Crystal Microbalance (QCM 200 purchased 
from Stanford Research Systems, SRS, USA), 
equipped with AT-cut quartz crystals (5 MHz) 
with optically flat polished titanium/gold 
electrodes on contact and liquid sides.  
MB release: The MB release from the 
multilayer coating on PET was evaluated by 
incubating the loaded film in 40 mL of PBS of a 
certain pH (2 or 7) and temperature (4 and 37 
ºC). The release medium was continuously 
stirred. At preset intervals, 0.25 mL supernatant 
was taken and 0.25 mL of fresh PBS was added. 
The sample solutions were analyzed using UV-
VIS spectroscopy (at 600 nm). In order to 
evaluate the MB release mechanism, the kinetic 
model of Korsmeyer & Peppas (1981) was 
applied. Non-linear regression analysis was 
performed using STATISTICA 7.0. 
 
RESULTS & CONCLUSIONS 
 
MB loading: The MB loading at different 
positions of the κ-carrageenan/chitosan 
multilayered film was evaluated by UV–Vis 
spectroscopy. The amount of MB loaded 
increased with the distance from the first layer. 
Similar results were obtained by Chung and 
Rubner (2002). The real-time adsorption of MB 
on κ-carragenan/chitosan pre-coated gold 
electrode surface was monitored using a QCM. 
It was found that MB loaded mass increased 
with the incubation time, exhibiting a very fast 
initial adsorption phase followed by a slower 
phase upon approaching a steady state. After 15 
min (time used for MB loading) the maximum 
adsorption of MB was guaranteed. In addition, it 
was observed that the MB had a higher 
adsorption in the sixth layer than in the other 
positions. 
 
MB release: The effects of temperature, pH and 
layer position on MB release were studied. It 
was observed that diffusional exponent values 
are within the limiting value of 0.5, suggesting 
that the MB release kinetics is Fickian in nature. 
Further, the values of correlation coefficient R
2
 
shows a fairly good fit of the model to the 
experimental data. It was observed that the MB 
position on the nanolayered film influences its 
release. At the same release conditions, the MB 
in the sixth layer enabled a fast release of this 
compound. Moreover, the rate of diffusion of 
MB from the κ-carrageenan/chitosan 
nanolayered film increased with increasing 
temperatures, from 4 to 37 °C. This behaviour 
can be explained by temperature effects on the 
solubility of diffusing molecules in films, on the 
nature of adhesive forces at interfaces and on 
molecular mobility (Vojdani and Torres 1990). 
The decrease of pH of the surrounding 
environment, from 7 to 2, also resulted in a 
faster MB release. At pH 2 the electrostatic 
interaction between κ-carrageenan and MB is 
very low, since the pKa value of the anionic 
sulfate groups on κ-carrageenan is around pH 2 
and at this pH the sulfate groups on the κ-
carrageenan molecules are protonated and 
consequently uncharged (Gu et al., 2005). 
Therefore, the pH decrease promotes the release 
of MB molecules from the nanolayered film. 
 
ACKNOWLEDGEMENTS 
 
The financial support from Fundação para a 
Ciência e Tecnologia (FCT) trough the PhD 
grant SFRH/BD/48120/2008 given to Ana C. 
Pinheiro is gratefully acknowledged.  
 
REFERENCES 
 
Bégin, A. and Van Calsteren, M.-R. 1999. 
International Journal of Biological 
Macromolecules, 26, 1, 63-67.  
Chung, A. J. and Rubner, M. F. 2002. 
Langmuir, 18, 1176-1183. 
Carneiro-da-Cunha, M. G., Cerqueira, M. A., 
Souza, B. W. S., Carvalho, S., Quintas, M. A. 
C., Teixeira, J. A. and Vicente, A. A. 2010. 
Carbohydrate Polymers, 82, 153–159. 
Gu, Y. S., Decker, E. A. and McClements, D. J. 
2005. Food Hydrocolloids, 19, 83–91. 
Jiang, B. and Li, B. 2009. International Journal 
of Nanomedicine, 4, 37–53. 
Korsmeyer, R. W. and Peppas, N. A. 1981. In: 
Controlled Release Delivery Systems, eds., 
Roseman, T. J. & Mansdorf, S. Z. Marcel 
Dekker, New York, 77–90. 
Vojdani F. and Torres, J. A. 1990. Journal Food 
Science, 55:841-846.  
Wang, X., Hu, X., Daley, A., Rabotyagova, O., 
Cebe, P. and Kaplan, D. L. 2007. Journal of 
Controlled Release 121, 190–199. 
 
AUTHOR BIOGRAPHY 
 
ANA C. PINHEIRO 
was born in Braga and 
went to the University of 
Minho, where she 
studied Biological 
Engineering and 
obtained her Integrated 
Master Degree in 
Biological Engineering 
in 2007. She worked for 
two years as a researcher in a project before 
start her PhD in Centre of Biological 
Engineering at University of Minho in 2009. 
Her e-mail address is: 
anapinheiro@deb.uminho.pt  
Her webpage can be found at: 
http://www.ceb.uminho.pt/pessoas/pid.aspx?id=
243 


English

Controlled release of a model bioactive compound from carrageenan-chitosan nanolayered films

Semana de Engenharia 2010 Guimarães, 11 a 15 de Outubro  CONTROLLED RELEASE OF A MODEL BIOACTIVE COMPOUND FROM -CARRAGEENAN/CHITOSAN NANOLAYERED FILMS  Ana C. Pinheiro, António A. Vicente Department of Biological Engineering E-mail: anapinheiro@deb.uminho.pt   KEYWORDS  Multilayers, layer-by-layer, transport modeling   ABSTRACT  Nanotechnology holds a great potential to generate very innovative solutions for the food industry and packaging is one of the many areas that can benefit from this new technology. The main goal of this work is to prepare nanofilms by layer-by-layer deposition using κ-carrageenan and chitosan, incorporate the model compound methylene blue and to evaluate its loading and release behaviour. The developed biodegradable nanofilms are a promising delivery system for application in food products, as a strategy for shelf-life extension.  INTRODUCTION  Layer-by-layer (LbL) assembly is based on the alternating deposition of oppositely charged polyelectrolytes and can be used to produce multilayers of nanometer thickness. Nanolayered films have promising applications on food products, as a strategy for shelf-life extension. However, there is a need to create multilayer systems that combine both biofuncionality and edibility. Chitosan, a natural cationic polysaccharide, is an excellent edible film component due to its good oxygen barrier properties and due to its intrinsic antimicrobial activity (Bégin & Van Calsteren, 1999). K-carrageenan, an anionic polysaccharide, is extensively used in food industry as gelling and stabilizing agent and has been reported as having excellent film forming properties (Park et al., 1996). In recent years, LbL nanolayered films have been investigated due to the possibility of controlling drug release (Wang et al., 2007). Furthermore, drug molecules can act as either functional drugs or components of the film. The release behaviour of bioactive compounds from nanolayered films depends on the permeability, on the disassembly or erosion of the multilayer structure and on other experimental variables (e.g. pH) (Jiang & Li, 2009). Methylene blue (MB), a monovalent cation of 374 Da, is a suitable model for loading and release experiments due to its large absorption peak in the visible range. MB has been used to investigate multilayer surface properties due to its ability to bind to available negatively charged functional groups (Chung & Rubner, 2002).  In this work multilayer nanofilms were prepared by LbL deposition using κ-carrageenan and chitosan onto a substrate (polyethylene terephthalate - PET). MB was incorporated in the nanolayered film and its loading and release behaviour was evaluated.  METHODS  Preparation of nanolayered film: The κ-carrageenan and the chitosan solutions were prepared at a concentration of 0.2 % (w/v) in distilled water and 1 % acid lactic, respectively. The solutions’ pH was adjusted to 7 and 3, respectively. MB solution was prepared at a concentration of 0.3 % (w/v) in 50 % (v/v) PBS and the pH was adjusted to 7. The nanolayered film was composed by an aminolyzed PET sustaining layer adsorbed with 5 layers (3 κ-carrageenan and 2 chitosan layers). The MB was incorporated in the film, forming the second, the fourth or the sixth layer, thus replacing the corresponding chitosan layer in those positions. The formation of nanolayered film was performed according to Carneiro-da-Cunha et al. (2010). MB loading: The loading of MB on each position of the multilayer structure was monitored by UV-VIS spectroscopy (Jasco 560, Germany). Real time deposition of MB on the coated electrode was recorded by a Quartz Crystal Microbalance (QCM 200 purchased from Stanford Research Systems, SRS, USA), equipped with AT-cut quartz crystals (5 MHz) with optically flat polished titanium/gold electrodes on contact and liquid sides.  MB release: The MB release from the multilayer coating on PET was evaluated by incubating the loaded film in 40 mL of PBS of a certain pH (2 or 7) and temperature (4 and 37 ºC). The release medium was continuously stirred. At preset intervals, 0.25 mL supernatant was taken and 0.25 mL of fresh PBS was added. The sample solutions were analyzed using UV-VIS spectroscopy (at 600 nm). In order to evaluate the MB release mechanism, the kinetic model of Korsmeyer & Peppas (1981) was applied. Non-linear regression analysis was performed using STATISTICA 7.0.  RESULTS & CONCLUSIONS  MB loading: The MB loading at different positions of the κ-carrageenan/chitosan multilayered film was evaluated by UV–Vis spectroscopy. The amount of MB loaded increased with the distance from the first layer. Similar results were obtained by Chung and Rubner (2002). The real-time adsorption of MB on κ-carragenan/chitosan pre-coated gold electrode surface was monitored using a QCM. It was found that MB loaded mass increased with the incubation time, exhibiting a very fast initial adsorption phase followed by a slower phase upon approaching a steady state. After 15 min (time used for MB loading) the maximum adsorption of MB was guaranteed. In addition, it was observed that the MB had a higher adsorption in the sixth layer than in the other positions.  MB release: The effects of temperature, pH and layer position on MB release were studied. It was observed that diffusional exponent values are within the limiting value of 0.5, suggesting that the MB release kinetics is Fickian in nature. Further, the values of correlation coefficient R2 shows a fairly good fit of the model to the experimental data. It was observed that the MB position on the nanolayered film influences its release. At the same release conditions, the MB in the sixth layer enabled a fast release of this compound. Moreover, the rate of diffusion of MB from the κ-carrageenan/chitosan nanolayered film increased with increasing temperatures, from 4 to 37 °C. This behaviour can be explained by temperature effects on the solubility of diffusing molecules in films, on the nature of adhesive forces at interfaces and on molecular mobility (Vojdani and Torres 1990). The decrease of pH of the surrounding environment, from 7 to 2, also resulted in a faster MB release. At pH 2 the electrostatic interaction between κ-carrageenan and MB is very low, since the pKa value of the anionic sulfate groups on κ-carrageenan is around pH 2 and at this pH the sulfate groups on the κ-carrageenan molecules are protonated and consequently uncharged (Gu et al., 2005). Therefore, the pH decrease promotes the release of MB molecules from the nanolayered film.  ACKNOWLEDGEMENTS  The financial support from Fundação para a Ciência e Tecnologia (FCT) trough the PhD grant SFRH/BD/48120/2008 given to Ana C. Pinheiro is gratefully acknowledged.   REFERENCES  Bégin, A. and Van Calsteren, M.-R. 1999. International Journal of Biological Macromolecules, 26, 1, 63-67.  Chung, A. J. and Rubner, M. F. 2002. Langmuir, 18, 1176-1183. Carneiro-da-Cunha, M. G., Cerqueira, M. A., Souza, B. W. S., Carvalho, S., Quintas, M. A. C., Teixeira, J. A. and Vicente, A. A. 2010. Carbohydrate Polymers, 82, 153–159. Gu, Y. S., Decker, E. A. and McClements, D. J. 2005. Food Hydrocolloids, 19, 83–91. Jiang, B. and Li, B. 2009. International Journal of Nanomedicine, 4, 37–53. Korsmeyer, R. W. and Peppas, N. A. 1981. In: Controlled Release Delivery Systems, eds., Roseman, T. J. & Mansdorf, S. Z. Marcel Dekker, New York, 77–90. Vojdani F. and Torres, J. A. 1990. Journal Food Science, 55:841-846.  Wang, X., Hu, X., Daley, A., Rabotyagova, O., Cebe, P. and Kaplan, D. L. 2007. Journal of Controlled Release 121, 190–199.  AUTHOR BIOGRAPHY  ANA C. PINHEIRO was born in Braga and went to the University of Minho, where she studied Biological Engineering and obtained her Integrated Master Degree in Biological Engineering in 2007. She worked for two years as a researcher in a project before start her PhD in Centre of Biological Engineering at University of Minho in 2009. Her e-mail address is: anapinheiro@deb.uminho.pt  Her webpage can be found at: http://www.ceb.uminho.pt/pessoas/pid.aspx?id=243 

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Controlled release of a model bioactive compound from carrageenan-chitosan nanolayered films

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