BSA and Fibrinogen Adsorption on Chitosa
Full Paper
BSA and Fibrinogen Adsorption on Chitosan/
k-Carrageenan Polyelectrolyte Complexes
Thiane N. Carneiro, Denise S. Novaes, Rodrigo B. Rabelo, Betul Celebi,
Pascale Chevallier, Diego Mantovani, Marisa M. Beppu, Rodrigo S. Vieira*
PECs of chitosan/k-carrageenan are prepared in three different volumetric rations. The
complex formation is characterized in order to evaluate the blending formation. Blood
compatibility is evaluated by protein adsorption (BSA and fibrinogen) and PEC toxicities
are determined with fibroblast cell viability
and proliferation. The swelling degree of PECs
decreases when the amount of chitosan
increases. Due to the linked film formation,
PECs decrease BSA adsorption and increase
fibrinogen adsorption when compared to the
pristine chitosan and k-carrageenan films.
Although pristine chitosan and k-carrageenan
films produced similar cell expansion and viability, the PEC 50:50 vol% chitosan/k-carrageenan
PEC may be acceptable as a new scaffold for
cell therapies, due to their effect on cell
survival.
1. Introduction
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Polyelectrolyte complexes (PECs) are formed by mixing
polysaccharides of opposite charges in an aqueous solution.[1] This network is formed by ionic interactions and is
characterized by a hydrophilic microenvironment with a
high water content and electrical charge density.[2] Such
complexes could be used as biomaterials for applications in
blood contact, such as the production of artificial hearts,
catheters, endovascular stents, hemodialysis membranes,
chemical sensors, and vascular implants. However, most
of these applications are limited by the thrombogenic
characteristics of polymeric surfaces, leading to the
requirement for anti-coagulant therapy by the patient.
Chitosan is a polycationic biopolymer, obtained by
alkaline deacetylation of chitin, which is the main
component of the exoskeleton of crustaceans.[2] This
wileyonlinelibrary.com
1
Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200482
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
R
T. N. Carneiro, D. S. Novaes, Prof. R. S. Vieira
Departamento de Engenharia Quımica, Universidade Federal do
, Centro de Tecnologia, Av. Mister Hull, s/n, Campus do Pici,
Ceara
Bloco 709 Pici, CEP 60455-760 Fortaleza, CE, Brazil
E-mail: rodrigo@gpsa.ufc.br
R. B. Rabelo, Prof. M. M. Beppu
Faculdade de Engenharia Quımica, Departamento de
^mica, Universidade Estadual de Campinas, Rua
Termofluidodina
Albert Einstein, FEQ, Bloco A, Barao Geraldo, CEP 13083-970
Campinas, SP, Brazil
Dr. B. Celebi, Dr. P. Chevallier, Prof. D. Mantovani
Laboratory for Biomaterials and Bioengineering, Department of
Min-Met-Materials Engineering, Canada Research Chair I in
Biomaterials and Bioengineering for the Innovation in Surgery,
Laval University & University Hospital Research Center, Quebec
City G1V 0A6, Canada
T. N. Carneiro et al.
www.mbs-journal.de
polysaccharide currently is receiving a great deal of
attention for use in medical and pharmaceutical applications, due to its favorable physicochemical and biological
properties, as this material is biocompatible, non-toxic and
antibacterial.[3] Films of chitosan also demonstrate interesting mechanical properties.[4] Chitosan is abundant in
nature and is also an excellent material for the development
of films, spheres, and fibers.[5] This material is insoluble in
water but becomes soluble when dissolved in acidic
solutions and it has great affinity for other biopolymers
with anionic properties in water. However, chitosan has
thrombogenic activity on its surface when in contact with
blood; its positive charges facilitate the adsorption of
plasma proteins such as albumin and fibrinogen at
physiological pH, promoting wound-healing.[2,6]
Over recent years, many studies have developed of
surface-modified chitosan for specific applications.[3,7–9]
The incorporation of negative charges can be an effective
way to reduce the thrombogenic properties of chitosan.
Hoven et al.[3] showed that surface modifications of chitosan
with sulfated groups containing a high negative surface
charge have lower amounts of adsorbed proteins than
pristine chitosan. However, the process to modify polymer
surfaces can involve specific materials there are expensive
and difficult to perform reactions. PECs of biopolymers with
different charges seem to be appropriate approaches to form
antithrombogenic surfaces. PECs of chitosan with different
polymers such as gelatin,[10] alginate, xanthan, pectin,
heparin, and dextran sulfate have been studied in literature
to be used in biomedical applications.[11]
Another polysaccharide of interest is carrageenan,
a linear biopolymer (alternating repeating units of
a-D-galactose and L-galactose) that is soluble in water and
extracted from red algae. Carrageenan is used in food and
pharmaceutical industries in gelling and stabilizing agents,
and for microencapsulation and immobilization of drugs
and enzymes.[12] The carrageenan structure has ester
sulfate groups on the galactose units, which are highly
charged groups that repel each other to keep an extended
and flexible configuration in the molecule. Depending on
the number and position of the ester sulfate groups on the
galactose units, the carrageenan isomer is known as k-, land i-carrageenan.[4] k-carrageenan has only one negative
group on its structure and presents good properties for
developing films. It is thought that negative charges (ester
sulfate groups) of the carrageenan contribute to the
formation of surfaces with antithrombogenic properties,
although such negative charges have a great influence on
the swelling properties of materials,[13] which is one of the
most important properties of films used for biomedical
applications.[14] Due to this charges, k-carrageen have a
high swelling ability, reason why it is interesting to form
PECs with other polymers that present low swelling
properties, in order to have a low swelling degree material.
The electrostatic attraction between cationic groups of
chitosan and anionic groups of k-carrageenan in solution
suggest the formation of a polyelectrolyte complex.
Chitosan/k-carrageenan PECs have been studied for many
biomedical applications, including tissue engineering,
cardiovascular devices and also drug delivery systems,
being the latter one the most reported application.[1,15–17]
Tapia et al.[17] evaluated the possibility of using chitosan/
carrageenan PECs as tablets for drug delivery. The swelling
behavior of carrageenan was responsible for the release of
the model drug. Spherical matrices of these polymers were
also prepared to promote the controlled delivery of theophylline.[18] Pinheiro et al.[19] showed that some drugs presented a
significant diffusion in chitosan/carrageenan complexes
nanolayers using polyethylene terephthalate as support.
The formation of PEC surfaces has the objective of
combining some of the characteristics of each polymer in
order to form a biocompatible material. The properties of
the PECs suggest that a wide set of characteristics of
polymers can be linked to constitute a desirable material.
Ideally, a biomaterial for blood applications should present
chitosan’s mechanical strength and the high negative
charge of carrageenan, resulting in a surface with
antithrombogenic characteristics. In this case, PECs have
the potential to form a biocompatible material that will
minimize adsorption of plasma proteins with adequate
chemical and mechanical properties, interaction with
metal surfaces, and appropriate swelling.
The aim of this study was to prepare and to characterize
composite films based on chitosan and k-carrageenan, by
different volumetric percentages, to conjugate the high
mechanical strength of chitosan and the antithrombogenic
properties of k-carrageenan. The PECs were characterized by
Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and degree of
swelling. The adsorption capacity of albumin (BSA) and
fibrinogen (FIB) and the albumin adsorption profiles were
analyzed at pH ¼ 7.4. Cell viability and proliferation were
also analyzed using fibroblasts on the PECs with pristine
films as controls.
2. Experimental Section
2.1. Materials
Chitosan with a deacetylation degree of 85% from Sigma (C3646
USA), k-carrageenan from Sigma (22048, USA), BSA from INLAB
(Brazil), and fibrinogen from Sigma (USA).
2.2. Preparation of Chitosan Films
Chitosan films were prepared by casting acidic chitosan solution
(2.0 wt%, prepared in 3.0 vol% acetic acid solution) and leaving to
Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200482
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
R
2
www.MaterialsViews.com
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
BSA and Fibrinogen Adsorption on Chitosan/k-Carrageenan Polyelectrolyte Complexes
www.mbs-journal.de
undergo the solvent evaporation process until the mass became
constant with time, following the method of Beppu and
Santana.[20] Subsequently, the films were immersed in a solution
of NaOH (1.0 mol L 1) for 24 h in order to neutralize the protonated
amino groups. The films were exhaustively washed with distilled
water until all acidic and basic residues were removed. Afterwards
the films were dried under vacuum for 2 d.
2.3. Preparation of k-Carrageenan Films
k-Carrageenan (2.0 wt% was dissolved in water under stirring at
80 8C for 1 h. The solution was spread into Petri dishes and kept at
60 8C until a constant weight was reached (6 h). The films were
immersed in a 2.0 mol L 1 KCl solution for 24 h and then washed
with distilled water. Afterwards the films were dried under
vacuum for 2 d.
2.4. Preparation of Chitosan/Carrageenan PEC Films
The chitosan solution (2.0 wt% in acetic acid 3.0 vol% was prepared
as described in preparation of chitosan films. k-Carrageenan was
added to KCl (0.1 mol L 1) with a concentration of 2 wt% and
stirred for 1 h for solubilization. These two solutions were mixed
under stirring with different volumetric proportions (25Chi/
75-Car, 50Chi/50-Car, and 75Chi/25-Car) for 24 h. Each solution
was then poured into a Petri dish (10 cm diameter) and dried at
60 8C until the mass became constant with time (5 h). The dried
films were placed in a NaOH aqueous solution (0.1 mol L 1) for
24 h. The films were dried under vacuum for 2 d at room
temperature.
300 W, using the Ka line of a standard (non-monochromatized) Al
(hn ¼ 1486.6 eV) and Mg (hn ¼ 1253.6 eV).
2.7. SEM
Surface morphology was studied by SEM analysis, using scanning
electron microscopy with an X-ray dispersive model Leo 440i
energy detector (Cambridge, England). Before being imaged by
SEM, the material was sputter-coater with a thin layer of gold
model SC7620, VG Microtech (Uckfield, England) with a thickness
of 92 Å.
2.8. Analysis by AFM
AFM imaging was performed using the tapping mode of a
Dimension TM 3100 Atomic Force Microscope (Veeco, Woodbury,
NY, USA). The tapping mode was applied using an etched silicon
tips (OTESPATM, tip radius
BSA and Fibrinogen Adsorption on Chitosan/
k-Carrageenan Polyelectrolyte Complexes
Thiane N. Carneiro, Denise S. Novaes, Rodrigo B. Rabelo, Betul Celebi,
Pascale Chevallier, Diego Mantovani, Marisa M. Beppu, Rodrigo S. Vieira*
PECs of chitosan/k-carrageenan are prepared in three different volumetric rations. The
complex formation is characterized in order to evaluate the blending formation. Blood
compatibility is evaluated by protein adsorption (BSA and fibrinogen) and PEC toxicities
are determined with fibroblast cell viability
and proliferation. The swelling degree of PECs
decreases when the amount of chitosan
increases. Due to the linked film formation,
PECs decrease BSA adsorption and increase
fibrinogen adsorption when compared to the
pristine chitosan and k-carrageenan films.
Although pristine chitosan and k-carrageenan
films produced similar cell expansion and viability, the PEC 50:50 vol% chitosan/k-carrageenan
PEC may be acceptable as a new scaffold for
cell therapies, due to their effect on cell
survival.
1. Introduction
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Polyelectrolyte complexes (PECs) are formed by mixing
polysaccharides of opposite charges in an aqueous solution.[1] This network is formed by ionic interactions and is
characterized by a hydrophilic microenvironment with a
high water content and electrical charge density.[2] Such
complexes could be used as biomaterials for applications in
blood contact, such as the production of artificial hearts,
catheters, endovascular stents, hemodialysis membranes,
chemical sensors, and vascular implants. However, most
of these applications are limited by the thrombogenic
characteristics of polymeric surfaces, leading to the
requirement for anti-coagulant therapy by the patient.
Chitosan is a polycationic biopolymer, obtained by
alkaline deacetylation of chitin, which is the main
component of the exoskeleton of crustaceans.[2] This
wileyonlinelibrary.com
1
Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200482
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
R
T. N. Carneiro, D. S. Novaes, Prof. R. S. Vieira
Departamento de Engenharia Quımica, Universidade Federal do
, Centro de Tecnologia, Av. Mister Hull, s/n, Campus do Pici,
Ceara
Bloco 709 Pici, CEP 60455-760 Fortaleza, CE, Brazil
E-mail: rodrigo@gpsa.ufc.br
R. B. Rabelo, Prof. M. M. Beppu
Faculdade de Engenharia Quımica, Departamento de
^mica, Universidade Estadual de Campinas, Rua
Termofluidodina
Albert Einstein, FEQ, Bloco A, Barao Geraldo, CEP 13083-970
Campinas, SP, Brazil
Dr. B. Celebi, Dr. P. Chevallier, Prof. D. Mantovani
Laboratory for Biomaterials and Bioengineering, Department of
Min-Met-Materials Engineering, Canada Research Chair I in
Biomaterials and Bioengineering for the Innovation in Surgery,
Laval University & University Hospital Research Center, Quebec
City G1V 0A6, Canada
T. N. Carneiro et al.
www.mbs-journal.de
polysaccharide currently is receiving a great deal of
attention for use in medical and pharmaceutical applications, due to its favorable physicochemical and biological
properties, as this material is biocompatible, non-toxic and
antibacterial.[3] Films of chitosan also demonstrate interesting mechanical properties.[4] Chitosan is abundant in
nature and is also an excellent material for the development
of films, spheres, and fibers.[5] This material is insoluble in
water but becomes soluble when dissolved in acidic
solutions and it has great affinity for other biopolymers
with anionic properties in water. However, chitosan has
thrombogenic activity on its surface when in contact with
blood; its positive charges facilitate the adsorption of
plasma proteins such as albumin and fibrinogen at
physiological pH, promoting wound-healing.[2,6]
Over recent years, many studies have developed of
surface-modified chitosan for specific applications.[3,7–9]
The incorporation of negative charges can be an effective
way to reduce the thrombogenic properties of chitosan.
Hoven et al.[3] showed that surface modifications of chitosan
with sulfated groups containing a high negative surface
charge have lower amounts of adsorbed proteins than
pristine chitosan. However, the process to modify polymer
surfaces can involve specific materials there are expensive
and difficult to perform reactions. PECs of biopolymers with
different charges seem to be appropriate approaches to form
antithrombogenic surfaces. PECs of chitosan with different
polymers such as gelatin,[10] alginate, xanthan, pectin,
heparin, and dextran sulfate have been studied in literature
to be used in biomedical applications.[11]
Another polysaccharide of interest is carrageenan,
a linear biopolymer (alternating repeating units of
a-D-galactose and L-galactose) that is soluble in water and
extracted from red algae. Carrageenan is used in food and
pharmaceutical industries in gelling and stabilizing agents,
and for microencapsulation and immobilization of drugs
and enzymes.[12] The carrageenan structure has ester
sulfate groups on the galactose units, which are highly
charged groups that repel each other to keep an extended
and flexible configuration in the molecule. Depending on
the number and position of the ester sulfate groups on the
galactose units, the carrageenan isomer is known as k-, land i-carrageenan.[4] k-carrageenan has only one negative
group on its structure and presents good properties for
developing films. It is thought that negative charges (ester
sulfate groups) of the carrageenan contribute to the
formation of surfaces with antithrombogenic properties,
although such negative charges have a great influence on
the swelling properties of materials,[13] which is one of the
most important properties of films used for biomedical
applications.[14] Due to this charges, k-carrageen have a
high swelling ability, reason why it is interesting to form
PECs with other polymers that present low swelling
properties, in order to have a low swelling degree material.
The electrostatic attraction between cationic groups of
chitosan and anionic groups of k-carrageenan in solution
suggest the formation of a polyelectrolyte complex.
Chitosan/k-carrageenan PECs have been studied for many
biomedical applications, including tissue engineering,
cardiovascular devices and also drug delivery systems,
being the latter one the most reported application.[1,15–17]
Tapia et al.[17] evaluated the possibility of using chitosan/
carrageenan PECs as tablets for drug delivery. The swelling
behavior of carrageenan was responsible for the release of
the model drug. Spherical matrices of these polymers were
also prepared to promote the controlled delivery of theophylline.[18] Pinheiro et al.[19] showed that some drugs presented a
significant diffusion in chitosan/carrageenan complexes
nanolayers using polyethylene terephthalate as support.
The formation of PEC surfaces has the objective of
combining some of the characteristics of each polymer in
order to form a biocompatible material. The properties of
the PECs suggest that a wide set of characteristics of
polymers can be linked to constitute a desirable material.
Ideally, a biomaterial for blood applications should present
chitosan’s mechanical strength and the high negative
charge of carrageenan, resulting in a surface with
antithrombogenic characteristics. In this case, PECs have
the potential to form a biocompatible material that will
minimize adsorption of plasma proteins with adequate
chemical and mechanical properties, interaction with
metal surfaces, and appropriate swelling.
The aim of this study was to prepare and to characterize
composite films based on chitosan and k-carrageenan, by
different volumetric percentages, to conjugate the high
mechanical strength of chitosan and the antithrombogenic
properties of k-carrageenan. The PECs were characterized by
Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and degree of
swelling. The adsorption capacity of albumin (BSA) and
fibrinogen (FIB) and the albumin adsorption profiles were
analyzed at pH ¼ 7.4. Cell viability and proliferation were
also analyzed using fibroblasts on the PECs with pristine
films as controls.
2. Experimental Section
2.1. Materials
Chitosan with a deacetylation degree of 85% from Sigma (C3646
USA), k-carrageenan from Sigma (22048, USA), BSA from INLAB
(Brazil), and fibrinogen from Sigma (USA).
2.2. Preparation of Chitosan Films
Chitosan films were prepared by casting acidic chitosan solution
(2.0 wt%, prepared in 3.0 vol% acetic acid solution) and leaving to
Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200482
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
R
2
www.MaterialsViews.com
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
BSA and Fibrinogen Adsorption on Chitosan/k-Carrageenan Polyelectrolyte Complexes
www.mbs-journal.de
undergo the solvent evaporation process until the mass became
constant with time, following the method of Beppu and
Santana.[20] Subsequently, the films were immersed in a solution
of NaOH (1.0 mol L 1) for 24 h in order to neutralize the protonated
amino groups. The films were exhaustively washed with distilled
water until all acidic and basic residues were removed. Afterwards
the films were dried under vacuum for 2 d.
2.3. Preparation of k-Carrageenan Films
k-Carrageenan (2.0 wt% was dissolved in water under stirring at
80 8C for 1 h. The solution was spread into Petri dishes and kept at
60 8C until a constant weight was reached (6 h). The films were
immersed in a 2.0 mol L 1 KCl solution for 24 h and then washed
with distilled water. Afterwards the films were dried under
vacuum for 2 d.
2.4. Preparation of Chitosan/Carrageenan PEC Films
The chitosan solution (2.0 wt% in acetic acid 3.0 vol% was prepared
as described in preparation of chitosan films. k-Carrageenan was
added to KCl (0.1 mol L 1) with a concentration of 2 wt% and
stirred for 1 h for solubilization. These two solutions were mixed
under stirring with different volumetric proportions (25Chi/
75-Car, 50Chi/50-Car, and 75Chi/25-Car) for 24 h. Each solution
was then poured into a Petri dish (10 cm diameter) and dried at
60 8C until the mass became constant with time (5 h). The dried
films were placed in a NaOH aqueous solution (0.1 mol L 1) for
24 h. The films were dried under vacuum for 2 d at room
temperature.
300 W, using the Ka line of a standard (non-monochromatized) Al
(hn ¼ 1486.6 eV) and Mg (hn ¼ 1253.6 eV).
2.7. SEM
Surface morphology was studied by SEM analysis, using scanning
electron microscopy with an X-ray dispersive model Leo 440i
energy detector (Cambridge, England). Before being imaged by
SEM, the material was sputter-coater with a thin layer of gold
model SC7620, VG Microtech (Uckfield, England) with a thickness
of 92 Å.
2.8. Analysis by AFM
AFM imaging was performed using the tapping mode of a
Dimension TM 3100 Atomic Force Microscope (Veeco, Woodbury,
NY, USA). The tapping mode was applied using an etched silicon
tips (OTESPATM, tip radius