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Carbohydrates Applications in Medicine, 2014: 1-29 ISBN: 978-81-308-0523-8 Editor: M. H. Gil

1. Polysaccharide-based polyelectrolyte

complexes and polyelectrolyte multilayers

for biomedical applications

P. Coimbra, P. Ferreira, P. Alves and M. H. Gil CIEPQPF, Department of Chemical Engineering, University of Coimbra, Polo II, Pinhal de Marrocos

3030-290 Coimbra, Portugal

1. Introduction

Polyelectrolytes (PELs) are polymers that bear innumerous ionizable

groups (1). When in solution, ionized PELs are able to form complexes with

other oppositely charged PELs, forming the so called polyelectrolyte

complexes (PECs). Depending on the formation conditions, and PELs

intrinsic properties, PECs can be formed with a different number of structures

and properties which grant, to the materials based on them, a wide range of

applications in different technological fields (e.g., medicine, paper-making

processes, water treatment, mineral separation, paint and food industries,

cosmetics and pharmacy) (1).

On the other hand, the alternating deposition of oppositely charged

polyelectrolytes on charged surfaces leads to the formation of thin

polyelectrolyte multilayer films (PEMs). This way of producing films,

designate by polyelectrolyte layer-by-layer (LbL) assembly, is a simple and

versatile method, that allows the construction of nanostructurated, functional

thin films with precise controllable properties (2-4).

Correspondence/Reprint request: Dr. Helena Gil, Chemical Engineering Department, Faculty of Science and

Technology, University of Coimbra, Coimbra, Portugal. E-mail: [email protected]

P. Coimbra et al. 2

Ionic polysaccharides, due to their polyelectrolytic nature, complex with

other natural or synthetic PELs, having the capacity, in this way, to form

PECs and PEMs materials. Since in general polysaccharides possess

excellent properties as biomaterials, such as non-toxicity, biocompatibility,

and bioresorbability (5), polysaccharides based PECs and PEMs are the

subject of much research in the biomedical and pharmaceutical fields. Drug

delivery, tissue engineering and modification/biofunctionalization of

biomedical devices surfaces are some of the main areas where

polysaccharides based PECs and PEMs find applications (5-11).

In this chapter, we will introduce the most important ionic

polysaccharides used until now to produced PECs and PEMs materials for

biomedical and pharmaceutical applications and highlight the main properties

exhibit by these types of materials. Furthermore, we present some selected

examples taken from literature that illustrate the different ways that

polysaccharides-based PECs and PEMs are being applied in the areas of

drug delivery, tissue engineering, and medical devices surface modification.

As framework, we start by introducing some basic concepts about PECs and

PEMs.

2. Polyelectrolyte complexes (PECs) and polyelectrolytes

multilayers (PEMs) formation and structure

2.1. Polyelectrolyte complexes (PECs)

PECs are formed in aqueous solutions by electrostatic interactions

established between charged domains of at least two oppositely charged

polyelectrolytes. Additionally, other interactions, such as hydrogen bonding,

van der Walls forces and hydrophobic and dipole interactions, can also

contribute to the complexation process (1). Fundamental research about PECs formation and structure have been

carried out since the 1960’s and the pioneering work o Michael et al (12, 13).

The great majority of these studies deals with PECs systems formed by

synthetic PELs, as for example poly(allylamine hydrochloride)/ poly(acrylic

acid) (14), and poly(styrenesulfonate)/ poly(diallyldimethylammonium) (15).

The general consensus among researchers is that PECs formations is a

entropy driven process: the complex formation implies the concomitant

release of the low molecular weight counterions initially bound to the ionic

groups of the polyelectrolytes, which drives the system into a higher level of

entropy (15, 16). This process is schematized in Fig. 1.

Ultimate PECs structure, composition, and physicochemical properties

are dictated by the characteristics the PELs precursors (e.g. chemical nature

PECs and PEMs for biomedical applications 3

Figure 1. Schematic representation of polyelectrolyte complex formation.

and position of the ionic sites, charge density, molecular weight, and chain

flexibility), mixing conditions (e.g. PELs concentration and charge ratio,

ionic strength and pH of the medium), and the preparation method (e. g.

mixing procedure).

The different structures that PECs can form, when in solution, are in

general categorized in three types (1, 17): i) water-soluble, ii) colloidally

stable, and iii) phase separated.

i) When polyelectrolytes with weak ionic groups and large differences in

molar mass are mixed in a non-stoichiometric ratio (ratio of cationic to

anionic functional groups) water-soluble aggregates at the molecular

scale can be formed (1, 18). Such structures consist of a long host

molecule sequentially complex with shorter guest polyions of opposite

charge. The formation and stability of these aggregates are dependent of

the presence and concentration of soluble salts and of the charge ratio of

the two PELs. By altering these parameters, water-soluble PECs can

form stable macroscopically homogeneous systems, coexist in solution

with insoluble, colloidally stable PECs, or aggregate and precipitate

(1, 18).

ii) PECs formation between polyelectrolytes of high and similar molecular

weight usually results in highly aggregated, macroscopic heterogeneous

systems. However, at low or moderate ionic strengths, and in extremely

diluted solution and non-stoichiometric charge ratio conditions, the

aggregation process can be stopped at a colloidal level, originating

colloidally stable PECs nanoparticles (1, 10, 16). In these conditions, the

PECs formation is mainly govern by the kinetics of the process, leading

to frozen structures, far from thermodynamic equilibrium (16). As so, the

final structure of these complexes depends strongly on the method of

P. Coimbra et al. 4

preparation, for example, the way and the order in which the polymers

are mixed (10, 16).

iii) When concentrated solutions of polyelectrolytes of high and similar

molecular weight are mixed in near stoichiometric rates, a two-phase

system is form, composed of a liquid phase and a PECs aggregates rich-

phase (1, 16). Depending on PELs properties and salt concentrations,

PECs-rich phase can exhibit rheological properties similar to the ones of

soft solids or, otherwise, have liquid-like properties, being call in this

case complex coacervates (16).

2.2. Polyelectrolytes multilayers (PEMs)

The preparation of polyelectrolytes multilayers (PEMs) films via the

layer-by-layer assembly (LbL) of oppositely charged polyelectrolytes on a

charged surface was first introduced by Decher and co-workers in 1992 (19).

Since then, this concept has generated a new and very active field of research.

Currently, PEMs are being investigated for innumerous applications in

different technological areas. Some of these applications include a different

range of photonic divices (20), fuel cells membranes, (21) and separation

membranes (22). Also, in the biomedical field PEMs are being extensively

investigated, mainly for applications as drug delivery systems and for coating

and biofunctionalization of biomedical devices (2-4, 23-27).

The PEMs formation via LbL is closely related to the process of PECs

formation in solution (28). Just like in PECs, the assembly process is

dominated by electrostatic interactions established between the oppositely

charged domains of PELs, although forces of other nature can contribute to

the formation and stabilization of the assemblies (29). Also,

thermodynamically this process is favored by the gain in entropy due to the

release of counterions.

In the LbL process, each polyelectrolyte layer is formed by the

absorption of charged polyelectrolytes from solution onto an oppositely

charged surface. The excess adsorption of the substances, i.e., charge

neutralization and resaturation, leads to a reversion of the surface charge,

which allows the deposition of an additional layer of a polyelectrolyte with a

charge opposite to that of the first one (24). This process is depicted in Fig. 2. One of the attractive features of the LbL method is its simplicity. In fact

the LbL strategy allows the construction of PEMs films without the need of

expensive or sophisticated equipment, using only simple procedures. The dip

coating method is the most simple and common way to LbL built-up. This

method consists in the sequential dipping of an object with a charged surface

in alternated aqueous solutions of oppositely charged polyelectrolytes.


Usually, a wash procedure is carried between each absorption step, in order

to remove uncomplexed polymer and assure more uniform layers (Fig. 2).

Other techniques already developed to produce PEMs by LbL include the

spin-coating method (30) and spraying LbL (31).

Just like in PECs, PEMs structure and properties are determined not only

by the intrinsic properties of the polyelectrolytes themselves but also by the

experimental conditions, namely the ionic strength and pH of the deposition

solutions.

Besides its appealing simplicity, the LbL assembly is regarded as a

extremely versatile bottom-up nanofrabication technique, since it allows the

construction of PEMs films with a great variety of architectures simply by

changing the types of polyelectrolyte components, the number of layers, and

the layering sequence (24). The LbL process is also viewed as being

applicable to virtually any pair of synthetic polyelectrolytes or charged

biopolymers such as ionic polysaccharides, proteins, enzymes, or nucleic acids.

Figure 2. Representation of PEMs formation via the layer-by-layer assembly method.

P. Coimbra et al. 6

Indeed, all these classes of materials have been already successfully used in

the construction of PEMs films (32). Also already proved possible is the

incorporation in PEMs films of charged nano-objects such as metal

nanoparticles (33), carbon nanotubes, and clay platelets (34), among others.

On the other hand, PEMs films can be assembled on surfaces of various

shapes and chemistries. Besides flat supports made of glass, metals or

polymers, PEMs films have been used to coat a variety of devices surfaces

such as micro-needles for transdermal drug delivery (35), stents (32) and

vascular prostheses (36), or even cryopreserved arteries (37).

Polyelectrolyte LbL assembly is also adaptable to the coating of small

particles, with micro or nano dimensions. By coating cores of colloidal

dimensions with alternated layers of polyelectrolytes, PEMs nano or

microcapsules, suitable as drug delivery vehicles, are formed (27, 38-42).

3. Ionic polysaccharides and modified ionic-polysaccharides

used in the preparations of PECs and PEMs for biomedical

applications

A large variety of ionic polysaccharides and modified ionic

polysaccharides have already been used to prepare PECs and PEMs films for

biomedical applications (5, 7, 11, 23). The chemical structures of the most

investigated until now are represented in Fig. 3 and 4. This group includes

polysaccharides like chitosan, alginate, pectin, the glycosaminoglycans

hyaluronan, heparin and chondroitin sulfate, and the modified

polysaccharides dextran sulfate and carboxymethylcellulose.

Chitosan, a random copolymer composed of -(1 4)-linked

2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose

units (Fig. 3), is undoubtedly the most used and investigated polysaccharide

for biomedical applications, particularly in the production of PECs and

PEMs biomaterials (5, 8, 11, 23). This ubiquitously is due to its numerous

Figure 3. Chemical structure of chitosan.


Figure 4. Chemical structures of the most important anionic polysaccharides used in

the preparation of PECs and PEMs for biomedical applications.

attractive properties, extensively reported in the literature, and to the fact that

chitosan is the only cationic polysaccharide easily available in large

quantities.

Chitosan is generally obtained by alkaline deacetylation of chitin, which

is the main component of the exoskeleton of crustaceans, such as shrimps. Its

P. Coimbra et al. 8

cationic character derives from the primary amino groups (NH2) present in

the desacetylated units. As the pka of chitosan’s amino groups is around 6-7,

in acidic conditions the majority of these groups become protonated (NH3+),

conferring to chitosan its polyelectrolyte properties and providing for its

solubility in aqueous solutions (5).

Since many polysaccharides have an anionic nature, there are a large

number of biocompatible anionic polysaccharides available to form PECs and

PEMs with chitosan or others cationic polyelectrolytes. These polysaccharides

are extracted from algae (such as alginate (5, 43)), plants (pectin (44, 45)) or

animal tissues (like hyaluronan, heparin, and chondroitin sulfate (25, 46)).

Also, some neutral polysaccharides (like dextran and cellulose), after extraction

can be chemical modified with the introduction of anionic groups originating,

in this way, semi-synthetic anionic polysaccharides, like dextran sulfate (47)

and carboxymethylcellulose (48). In the great majority, these anionic

polysaccharides bear in their structures carboxylic groups (COO-), with a pka

around 3 – 5, or sulfonate groups (RSO3-), with a pka around 0.5 – 1.5 (23).

Alginate (Fig.4) is a natural occurring anionic polysaccharide typically

obtained from brown algae by alkaline extraction (5, 43). Alginate comprises

a whole family of linear copolymers containing blocks of (1,4)-linked -D-

mannuronate (M) and -L-guluronate (G) residues. The blocks are composed

of consecutive G residues (GGGGGG), consecutive M residues

(MMMMMM), and alternating M and G residues (GMGMGM). Alginates

extracted from different sources differ in M and G contents as well as the

length of each block.

Alginate has been extensively investigated and used for many biomedical

applications, due to its biocompatibility, low toxicity, relatively low cost, and

the capacity to form gels in the presence of divalent cations such as Ca2+

(5, 43).

Pectins are a family of complex polysaccharides found in the cell walls

of higher plants. They are composed by D-galacturonic acid residues and a

range of neutral sugars such as rhamanose, galactose, arabinose and lesser

amounts of others. These components are arranged in linear and branched

structures (5, 10, 44, 45). Commercial pectin extracts, obtain essentially from

apple pomace and citrus peel through an aqueous acid extraction process,

consists mainly of linearly 1,4-linked- -D-galactosyluronic acid residues,

which may be methyl-esterified at varying extents (Fig. 4). Like alginate,

pectins with a degree of methyl esterification inferior to 50% form gels in the

present of divalent cations like Ca2+

. Pectin has a long safe history of use in the food processing industry,

where it is utilized as gelling, stabilizing and thickening agent (44). Pectin is


also recognized as an important soluble fiber in human diet and there are

clear evidences that its consumption is beneficial to health. For example,

pectins have the capacity to lower the blood cholesterol level and to slow

down the absorption of glucose (44, 45). Pectin has also being regard with

increasing interest for pharmaceutical and biomedical applications (49, 50).

The polysaccharides hyaluronan (HA), chondroitin sulfate (CS), heparin

(HEP) and heparan sulfate (HS) are glycosaminoglycans (GAGs), a very

important group of anionic polysaccharides present in the extracellular

matrix of all animal tissues, where they interact and bind with a wide range of

proteins (like chemokines, cytokines, growth factors, morphogens, enzymes

and adhesion molecules) participating and regulating, in this way, a variety of

biological processes such as cell adhesion, cell growth and differentiation,

cell signaling, or blood coagulation (46). GAGs are linear polymers with

disaccharide repeating units composed of one uronic acid (D-glucuronic

acid or L-iduronic acid) and one amino sugar (D-galactosamine or

D-glucosamine). All GAGs are fully charged polyanions at physiological

conditions, containing carboxyl and/or sulfonate groups. Thus, GAGs

electrostatic characteristics, such charge density and distribution, determine

not only their physical-chemistry but also the interactions established with all

the other molecules and macromolecules present in the extracellular matrix

and, in consequence, GAGs biochemical functions.

Hyaluronan (HA), or hyaluronic acid, is the simplest and the only non-

sulfated GAG, compose of the repeating disaccharide β-(1,4)-D-glucuronic

acid–β(-1,3)-N-acetyl-D-glucosamine (46, 51-53) (Fig. 4). HA is also known

to be the only GAG that, in vivo, is not covalently linked to a core protein

(46). In the human body, HA occurs in the salt hyaluronate form and it is

present in almost all biological fluids and tissues, being found in high

concentrations in the skin, umbilical cord, vitreous humor and synovial fluid.

With a high moisture retention ability and viscoelasticity, HA performs

important mechanical and structural functions such as space filler, lubrication

of the joints, wetting agent, flow barrier within the synovium and protector of

cartilage surfaces (46). Additionally, HA has many important functional

roles, including signaling activity during embryonic morphogenesis,

pulmonary and vascular diseases, and wound healing (46, 52). HA is also

associated with cancer invasiveness and metastasis (54).

HA is used in many medical and pharmaceutical applications. For

example, it is used as a diagnostic marker for many diseases including

cancer, rheumatoid arthritis and liver pathologies, as well as for

supplementation of impaired synovial fluid in arthritic patients by means of

intra-articular injections. It is also commonly used in many ophthalmological,

otolaryngological and cosmetic surgeries (52). HA has been extensively

P. Coimbra et al. 10

investigated by biomaterial scientists. Many types and forms of HA based

materials, often obtained by the chemical modifications of this polysaccharide,

have already been produced for applications in tissue engineering, drug

delivery and wound healing (51). Traditionally extracted from rooster combs,

HA is, nowadays, mainly produced via microbial fermentation, with lower

production costs and less environmental pollution (53).

Chondroitin sulfate (CS) is the most abundant and prevalent GAG, being

widely distributed in humans, other mammals, and invertebrates (55). CS is

comprised of the disaccharide [-4-GlcA-β1-3-GalNAc-β1-] (where GlcA, is

the monosaccaride glucuronic acid and GalNAc is N-acetyl galactosamine).

In vivo, and during polymerization, CS suffers sulfation of some OH groups

(OH groups on the C4 and C6 of GalNAc and C2 and C3 of GlcA) through

the activity of a variety of sulfotransferases, resulting in a polymer containing

O-sulfo groups at various positions (Fig.4) (55-57). According to their

sulfation pattern, chondroitin sulfates are classified in various classes (Figure 4).

In vertebrates, the CS found in more quantity are CS type A (most commonly

sulfated (90%) at the C4-position of GalNAc) and CS type C (most commonly

sulfated (90%) at the C6-position of GalNAc) (57). Differences in sulfation

pattern are known to deliver diverse, and possibly conflicting, outcomes, both

in in vitro and in in vivo studies, which evidentiates that different CS have

distinctive biological activity (3, 56, 58). Therefore, currently, the

investigations focused on CS bioactivity accurately describe the structural

characteristics of the CS used (57, 58).

Contrary to hyaluronan, commercial CS is still currently obtained from

animal tissues, such as bovine trachea, pig nasal septa, chicken keel, shark

fins, and fish cartilage. In this way, CS extracts are usually a mixture of

different CS types (56).

In Europe and other countries, CS is classified as a drug, being approved

for the treatment of osteoarthritis. In the United States, CS is regulated by the

FDA as a dietary supplement, being commercialized, often in combination

with glucosamide, as a nutraceutical (55, 56). Since nutraceutical products

are not strictly regulated as their pharmaceutical counterpartners, this kind of

products containing CS are seen by the scientific community with some

skepticism and concern, since sometimes they present low-quality, are

ineffective, and can be even potentially dangerous (55, 56).

Heparin and heparan sulfate are the most complex GAGs, consisting of at

least ten different monosaccharide building blocks that can be combined into

a large number of different disaccharide sequences (Fig.4) (46, 59). Heparin

(HEP) is a highly sulfated GAG, containing in average 2.5 sulfate groups per

disaccharide (60), which makes heparin one of the most acidic


macromolecules in nature. Heparan sulfate (HS) is structurally similar to

heparin, but possesses regions that are much less sulfated.

The uronic acid of heparin and heparin sulfate may be either α-L-iduronic

(IdoA) or β-D-glucuronic acid (GlcA) and can be unsubstituted or sulfonated

at the 2-O position. The glucosamine residue may be unmodified (GlcN),

N-sulfonated (GlcNS) or N-acetylated (GlcNA), and can contain variable

patterns of O-sulfonation at the 3-O and 6-O positions (46, 59, 61).

In heparin, the most frequent disaccharide repeating unit is composed of

2-O-sulfated L-iduronic acid (IdoA2S) (1,4)-linked to a 6-O-sulfated, N-

sulfated D-glucosamine (GlcNS6S), while in heparin sulfate the most common

repeating unit is formed by the non-sulfated disaccharide composed of

D-glucuronic acid (GlcA) (1,4)-linked to N-acetylglucosamide (GlcNAc)

(46, 59).

Heparin is known to be highly evolutionarily conserved, with similar

structures found in a broad range of vertebrate and invertebrate organisms

(46). While HEP is synthesized by and stored exclusively in mast cells, HS is

expressed on cell surfaces, being an integral part of the extracellular matrix

(46, 61).

HEP and HS possess anticoagulant activity, being commonly used in the

prevention and treatment of many thromboembolic disorders. Heparin is also

used to coat the surfaces of blood-contacting medical devices, in order to

improve their hemocompatibility (62).

Much research on the structure, function, and biological activity of

heparin and heparin sulfate has been carried out, many dedicated to elucidate

the mechanism thought which these GAGs prevent blood coagulation.

Evidences suggest that heparin binds to enzyme inhibitor antithrombin III

(AT), activating this molecule, that in turns, inactivates thrombin and other

proteases involved in blood clotting (46, 59, 61).

Just like chondroitin sulfate, heparin is obtained from animal tissues,

mainly from porcine intestinal mucosa (46, 61).

Dextran sulfate (Fig. 4) is an anionic synthetic derivative of dextran, a

neutral polysaccharide produced by certain bacteria strains. Dextrans are

homopolymers formed by an -(1 6) linked D-glucose main chain branched

with side chains of D-glucose residues connected, in the majority, by -(1 6)

and -(1 3) glycoside bonds (47).

Dextran sulfate (DS), usually commercialized in the form of sodium salt,

is prepared by the sulfation of dextran (47). Typically, it has a substitution

degree of around two, which means that each glucose unit bears, in average,

two sulfate groups, located normally at C2 and C4 position. DS is consider a

synthetic mimic of heparin and heparin sulfate exhibiting, like these GAGs, a


broad spectrum of biological activities, including anti-coagulant activity (47).

Inclusively DS has already been investigated as potential substitute for

heparin in anticoagulant therapy. However its anticoagulant activity was

found to be considerably lower than heparin. Nevertheless, and because DS is

a lot cheaper than GAGs, DS is continued to be investigated as substitute for

heparin and heparan sulfate in other specific applications (47).

Carboxymethylcellulose (CMC) (Fig. 4), usually available in the form of

a sodium salt, is an anionic synthetic derivative of cellulose, the most

abundant naturally occurring polysaccharide, consisting of a linear chain of

several hundred to over ten thousand β(1→4) linked D-glucose units.

Industrially, CMC is generally produced by the etherification of alkali-

cellulose with sodium monochloroacetic acid (48). The functional properties

of CMC depend on the degree of substitution (number of carboxymethyl

groups per anhydroglucose unit) as well as on the chain length of the

cellulose backbone structure. By varying these two parameters, a broad

number of CMC grades are manufactured, with a wide range of properties,

tailored for specifically applications. Due to its low commercial cost and

many attractive properties, which include unique rheological properties,

CMC is widely use in many industrial fields as a stabilizing, thickening, film-

forming and binding agent. Since CMC is non-toxic, this cellulose derivative

is also extensively applied in the pharmaceutical and cosmetic industries,

being present in the composition of the products such as tablets, syrups,

suspensions, ointments, creams, toothpastes, and many other products (48).

CMC alone, or in combination with other natural or synthetic polymers, has

been used to prepare hydrogels for drug delivery and other biomedical

applications (63). Because of the polyelectrolyte nature of this polymer,

CMC-based hydrogels usually exhibit a pH and ionic strength-dependent

swelling behavior, which make them attractive for some specific applications. Besides the polysaccharides introduced above, which are the most used

until now, in literature there are several examples of others anionic

polysaccharides that already have been mixed with chitosan to form PECs

and PEMs materials, such as carrageenan (64), xanthan gum (65), gum

kondagogu (66), and carboxymethyl starch (67).

As an alternative to chitosan, anionic polysaccharides have been used

in combination with the cationic polypeptide poly(L-lysine) (68-70),

the synthetic PELs poly(ethylene imine) (PEI) (71) and poly(allylamine-

hydrochloride) (PAH) (72) or even with biodegradable cationic poly( -amino

esters) (73, 74). On the other hand, chitosan can also be used in combination

with other PELs, such as polyacrylic acid (75) polystyrene sulfonate (PSS)

(76), or the anionic polypeptide poly(L-glutamic acid) (77).


4. Applications of polysaccharide-based PECs and PEMs in

the biomedical field

An attractive aspect of PECs and PEM is the simplicity of their preparation

process. In fact, these materials are formed in exclusively aqueous

environments, at room temperature, and without the need of organic solvents or

other potential toxic chemicals like initiators, catalysts or chemical crosslinkers.

Due to this, they usually exhibit an excellent biocompatibility.

Additionally, and in the case of polysaccharides based PECs and PEMs,

they preserve the attractive properties displayed by the individual

polysaccharides constituents, such as biocompatibility, biodegradability and

biological activity.

On the other side, the use of polysaccharides presents certain limitations

and disadvantages compared to their synthetic counter partners. One of the

greatest drawbacks is the inherent variability of theirs properties. Most

polysaccharides are extracted from natural tissues and are, thus, dependent on

natural variations. Further, the quality of the materials can vary widely from

one producer to another, due to the different extractions conditions and

purifications steps employed (23). In addition, polysaccharides present also

an elevate polydispersity. This variability difficults the productions of PECs

and PEMs material with batch-to-batch reproducibility.

Ionic polysaccharides, with the exception of dextran sulfate (10) and

sometimes heparin and chondroitin sulfate (60), are weak polyelectrolytes.

This means that the degree of ionization of these polysaccharides is pH-

dependent. Due to this, the pH range that allows the formations of PECs and

PEMs usually lies between the pka values of the two polysaccarides.

Polysaccharide PECs and PEMs materials are highly hydrophilic, with

elevated swelling capacity that is, usually, pH dependent. These materials are

normally fragile in the hydrate state, and can even dissolve rapidly. Thus, and

in order to reinforce then, sometimes they are further crosslinked by the

addition of covalent crosslinks, using reagents like glutaraldehyde (78) and

water soluble carbodiimides (68). However, the additions of chemical

crosslinkers may decrease the biocompatibility (11).

4.1. Drug delivery

4.1.1. Polysaccharide complexes nanoparticles and other PECs matrices

Polysaccharide PECs drug delivery systems can assume the form of: i)

nanoparticles, when the mixing conditions of the two charged


polysaccharides originates a colloidally stable system, or ii) macroscopical

matrices, when the mixing of the two PELs originates a two-phase system.

i) The polyelectrolyte complexation of two opposite charged PELs in

solution can originate colloidally stable polyelectrolyte complexes

nanoparticles (PCNs) (9, 10, 25). Contrary to other nanoparticles formations

methods, this is a rather simple procedure that consists, basically, in the

mixing of the two PELs solutions. Due to this straightforwardness,

polysaccharide based PCNs have been investigated as possible drug carriers,

especially of labile drugs, such as therapeutic proteins and genes, since the

process of PCNs formations is carried out in mild conditions and in a

exclusively aqueous medium, conditions that favor the preservation of the

biological activity of these fragile drugs (17).

In general, the formation of stable PCNs requires the use of extremely

diluted solutions and non-stoichiometric ratios of the two constituents (1, 10).

PELs intrinsic properties (such as charge ratio and molar mass), the charge

ratio of the two oppositely charged PELs, concentrations, order and rate of

mixing, presence of salt and the pH of the medium, are all experimental

parameters that, not only determine the colloidal stability of the formed PCNs

but also important properties such as composition, size distribution and zeta

potential (10, 17). As so, the development of PCNs delivery system involves

the investigation and optimization of the referred experimental variables, in

order to obtain stable PCNs in biological conditions and which,

simultaneously, are able to function as efficient drug delivery systems (i.e.,

with high loading capability and desired release profile, ability to protect the

therapeutics of degradation in biological environment, and capacity to target

the drug to the desired organ or tissue).

The nanoparticles formed by the direct complexation of chitosan or its

derivatives with genetic material, for gene delivery applications, are the most

investigated and recognized type of polysaccharide PCNs based delivery

systems (79). In this case, the genetic material (DNA, RNA, or other type) is

simultaneously the anionic constituent of the PCNs and the therapeutic

molecule. These types of systems are reviewed in another chapter of this book.

Several polysaccharides based PCNs have been developed for the delivery

of therapeutics such as vaccines, growth factors, and other proteins. The PCNs

formed by the complexation of chitosan and dextran sulfate (CHIT/DS) are one

of the most used systems for this purpose. The formation process and properties

of CHIT/DS PCNs have been thoroughly investigated by several authors (10,

80). Delair has recently review this knowledge and the drug delivery

applications of this system (10). Examples of bioactive molecules already

incorporate in CHIT/DS PCNs are the fibroblast growth factor-10 (FGF)(81),

insulin (82), the model protein BSA (7) and the antigen protein p24 (6).


Besides genes and protein, polysaccharide PCNs have also been investigated for the delivery of low molecular weight drugs such as chemotherapeutics and antibiotics. For example, Motwani et al developed chitosan/alginate mucoadhesive PCNs for the prolonged topical ophthalmic delivery of the antibiotic gatifloxacin, using a response surface methodology to optimize some of the PCNs properties such as size, zeta potential, drug loading, and burst release (83). Doxorubicin (DOX), a drug with significant anti-tumor activity, was also formulated in different polysaccharide PCNs delivery systems (84-86). Although DOX is commonly used in the treatment of a wide range of cancers, its clinical application suffers from a few inherent risks, due to the serious side effects frequently reported. This justifies the great efforts done in the persecution of efficient delivery systems for this drug. Tan et al developed a DOX delivery system based on CHIT/DS PCNs and tested in vivo, in mice bearing orthotopic osteosarcomas (86). Tumors treated with DOX CHIT/DS PCNs, administrated locally, decreased in volume. Other group incorporated DOX in chitosan/chondroitin sulfate PCNs and investigated different strategies to improve the efficiency of the drug delivery system: the chemical crosslinking of the formed PCNs, in order to enhance the stability of the nanocarriers in the presence of serum proteins (85), and PCNs funtionalization with folic acid, in order to actively target the delivery system to the cancer cells (87). ii) When the mixing of two PELs originates a two-phase PECs system, the PECs rich-phase can be isolated, washed, dried and processed in a variety of forms. Depending on the drying method (air drying, freeze-drying, spray-drying) and/or further processing, polysaccharide based PECs can be obtained on the form of films (75), sponges (84, 88, 89), particles (66, 90), tablets (66, 67, 91-93), and so on. These PECs materials are usually very hydrophilic and are frequently classified as physically cross-linked hydrogels (11). All these different type of polysaccharide based PECs matrices have

already been used to prepare drug delivery systems. The drug can be

incorporated in these matrices during PECs formations (66, 88, 90), after

PECs isolations and before drying (89), or simply by physically mixing the

solid drug with dry PECs granules and later process the mixture into tablets

(67, 92, 93). In nearly all works, chitosan is used as the polycation, combined

with diverse anionic polysaccharides, namely with alginate (84, 88, 90) and

pectin (89, 92). Alternatively, chitosan has also been used in combination

with poly(acrylic acid) (75) and other synthetic acrylic-based polymers (93).

Polysaccharide–based PECs delivery systems have been proposed for many applications. Two of them are as wound dressing materials with controlled drug release abilities (84, 88, 94) and as matrix for colon-specific drug delivery (67, 90-93).


4.1.2. PEMS micro/nanocapsules

PEMs micro/nanocapsules formed by layer-by-layer deposition of

opposite charged polyelectrolytes on the surface of template particles with

colloidal dimensions are regarded as interesting and versatile drug delivery

vehicles. The diverse applications of this type of constructs in the field

of drug delivery have been extendedly review in a series of recent articles

(27, 38, 40-42, 95).

The preparation of these capsules is done by submitting the micro/

nanoparticles templates to successive cycles of suspension, centrifugation and

resuspension in the polyelectrolyte solutions. The therapeutics can be

incorporated in the capsules by a number of techniques that can be divided in

three categories, depicted in Fig. 5 (40): the post-loading approach; the

encapsulation of crystalline drug particles; and the incorporation in porous

template particles before capsule formation.

In the post-loading approach the molecules of interest are loaded into

pre-formed capsules (Fig. 5A). This is usually done by inducing a reversible

Figure 5. Different methods for encapsulating therapeutics in capsules fabricated by

the layer-by-layer assembly of polyelectrolytes. A) loading preformed capsules; B)

encapsulation of crystalline therapeutic particles; C) incorporation in porous template

particles before capsule formation (reprinted with permission from Johnston et al. (40).


change in the permeability of the capsule wall which allows the therapeutic to

diffuse into the capsules. This permeability can be induced by varying salt

concentration, pH, or temperature (77, 96, 97). Other technique of

encapsulation consists in adsorbing the therapeutic in a porous particle

template that can be subsequently LbL coated with PEMs (Fig. 5C). After

capsule formation, the cores can be removed by dissolution, yielding a

hollow capsule loaded with the therapeutic (39, 98). Alternatively,

nanoparticles of water insoluble or poorly soluble drugs can be directly

coated with a PEMs shell (Fig. 5B), in order to formed stable aqueous

nanocarriers with controllable drug release capability (99-102).

LbL PEMs micro/nanocapsules made exclusively of polysaccharides or

with one polysaccharide component have already been prepared and

investigated as drug delivery vehicles of different types of therapeutics such

as DNA (98), proteins, (39, 96) anti-cancer drugs (77, 97, 103, 104), and

other low molecular weight drugs (77, 101, 102).

For example, Ye et al prepared PEMs microcapsules by the layer-by-

layer assembly of chitosan and sodium alginate on melamine formaldehyde

template microparticles followed by removal of the templates through

dissolution at low pH. Insulin was then loaded into the microcapsules through

altering permeability of the capsule membrane by varying the pH of the

medium and the temperature (96). Alternatively, Itoh et al used a pre-loading

strategy to encapsulate the model protein albumin in biodegradable hollow

capsules made by LbL assembly of chitosan and dextran sulfate (105). Before

capsule formation, the model protein was absorbed into mesoporous silica

microparticles and after capsule assembly the silica template core was removed

by dissolution with hydrofluoric acid. Sustained release of the encapsulated

proteins was attained due to the enzymatic degradation of the hollow capsules.

Finally, in literature there are also several examples of poor water soluble drug

micro/nanoparticles coated with polysaccharide PEMs shells for controlled

delivery applications. Specifically, several non-steroidal anti-inflammatory

drugs like ibuprofen (101), indomethacin (99, 100) and naproxen (102).

4.2. Surface modification and biofunctionalization of biomedical

devices

The bulk and surface properties of a material are the key properties for a

successful medical device. This issue has been a challenge for bioengineers

in the fields of tissue engineering, biomaterials and biophysics.

Mechanical properties are the key properties to be evaluated in a

biomedical device, however, a material with the perfect bulk properties might

not be suitable for a desired application due to its surface properties. The


surface mediates whether a material is recognized or not as a foreigner

device. The surface of a material works as the interface between the material

and the host tissue; therefore it is capable to initiate a wide variety of

processes, from the initial inflammatory reaction to rejection or failure of the

device.

In order to overcome these issues, scientist have been gathering efforts

towards tailoring the surface of materials, specially of those used in

biomedical applications, namely, metals, polymers and ceramics. The surface

of these materials are normally functionalized in order to trigger a specific

cell response, in other words, in order to become bioactive (2). Among all

coatings, polymers and biopolymers coatings are the most versatile, offering

a wide range of opportunities to tuning the materials chemical and physical

properties. In this way, polymeric coatings are widely use in the surface

modification of biomedical devices.

Natural biopolymers, due to their similarity to human tissues are

advantageous as biomimetic coatings. As previously mentioned,

polysaccharides have several advantageous characteristics such as low

toxicity, biocompatibility, stability, low cost, hydrophilic nature and

availability of reactive sites, such as free carboxyl and hydroxyl groups,

which can be used for chemical modification (105). In order to tailor a

surface using polysaccharides, techniques such as oxidation, esterification,

amidation, or grafting methods can be used (106). Among these techniques,

the ones that have shown to be useful for saccharide chemistry in surface

modification are: the covalent attachment of self-assembled monolayers

(SAMs), formation of polymer brushes, and the layer-by-layer (LbL)

assembly of polyelectrolyte multilayers (PEMs).

The formation of PEMs is the more common strategy for modifying

surfaces with polysaccharides. This method has been widely used in several

nanotechnology applications, mainly to modify cell-surface and protein-

surface interactions, to encapsulate cells, and to stabilize and deliver

biologically active proteins (25)

The LbL assembly technique of polysaccharides coatings on surfaces

allowed to understand their interactions with proteins, biological fluids, and

cells. Therefore, surfaces coated with very hydrophilic polysaccharides,

such as hyaluronic acid, showed to be resistant to protein adsorption and

cell adhesion (107). Surfaces coated with sulfated polysaccharides have the

ability to reduce platelet activation and have anticoagulant activity (108).

Nevertheless, polysaccharide PEMs can also be used to enhance cell

adhesion and control cell function, such as chitosan-heparin PEMs. These

PEMs were used to coat stents in order to improve the endothelialization

(109).


Other application of the LBL technique is the coating of scaffolds. Zhu

and coworker (110) introduce free amino groups on the surface of polyester

scaffolds by reacting the ester groups with diamine. Afterwards, using the

LbL technique, layers of negatively charged poly(styrene sulfonate sodium

salt) and positively charged chitosan were alternately deposited onto the

positively charged amino groups of the surface. This method showed that the

outer chitosan layer significantly improved biocompatibility of poly(L-lactic

acid) based scaffolds.

4.3. Tissue engineering

The term Tissue engineering (TE) was initially defined by the attendees

of the first NSF (National Science Foundation, USA) sponsored meeting in

1988 as a research field that relies in the “application of the principles and

methods of engineering and life sciences towards the fundamental

understanding of structure-function relationships in normal and pathological

mammalian tissues and the development of biological substitutes that restore,

maintain or improve tissue function” (111). Or, in other words, TE being

explored in an attempt to repair or replace portions of and/or entire tissues. In

fact, the main goal of such studies is to overcome the restrictions associated

with processes such as organ donation and transplantation. With the

possibility of creating new tissues or even organs from the cells of the

patient, which means that donor and receptor are in fact the same individual,

problems like donor shortage and immunogenicity are avoided. Therefore,

supplementary therapies, like immunosuppression, will no longer be

necessary. As a result, patients will face a much easier and less risky recovery

and costs associated with treatment will be considerably lower (112).

Independently of which tissue is to be engineered, the basic principles of

TE remain the same. TE takes advantage of using biomaterials as scaffolds

intended to act as three dimensional (3D) structures capable of supporting

cell growth and differentiation, as well as promoting cell adhesion and

migration. Several stages are involved in the process (113) starting with cell

sourcing, isolation and proliferation, preparation of biodegradable 3D

scaffolds, seeding of cells on those scaffolds, in vitro culture in a bioreactor

or incubator and finally implantation in the area of defect in patient’s body

(Fig. 6).

The major concern when designing scaffolds for tissue engineering is the

selection of suitable materials. There are a few general requirements that all

scaffolds, independently of the tissue to be engineered, should meet. First of

all, they should be biocompatible, which means that no immune response

should be triggered by their presence in the organism (114). They should also


Figure 6. Tissue engineering and its principles.

present a highly porous structure which allows cell in-growth, permeation of

nutrients and metabolites and in vascularized tissues, the growth of new

blood vessels (angiogenesis) (115). The matrix should also be biodegradable

and finally should present a similar architecture as the extracellular matrix

(ECM) (116).

Scaffolds may be prepared in a wide variety of morphologies, namely

fibers, capsules, spheres, sponges and hydrogels (Fig. 7). Their composition

and properties may be modified and therefore adjusted for the desired

application.

These scaffolds may also be used as substrates for the delivery of

bioactive species and can be prepared from different biocompatible and/or

biodegradable materials that include both natural and synthetic polymers.

Natural materials, namely polysaccharides, have been extensively used in

the production of scaffolds for tissue engineering since they are similar to the


Figure 7. Possible morphologies presented by scaffolds for tissue engineering.

extracellular matrix of natural tissues. Several different polyelectrolyte

complexes between oppositely charged polysaccharides have been prepared

(117) and tested as possible scaffolds for different tissues regeneration. Three

main morphologies have proved suitable to be applied as matrices for tissue

regeneration: fiber meshes, sponges and PEMs films.

4.3.1. Fiber meshes

As previously mentioned, most of the cells in multicellular organisms are

embedded in a complex and organized meshwork of macromolecules,

(proteins and polysaccharides), that compose the extracellular matrix (ECM).

This means that for cells, this is their normal environment in which they live,

grow and divide. One of the challenges for researchers in the area of TE is to

reproduce such environment by designing polymeric scaffolds with

mechanical and biological properties similar to the ones of EMC (118). For

these reasons, the preparation of scaffolds under the form of fibers comes as a

natural way of reproducing their natural surroundings.


Numerous techniques have so far been used in the preparation of fibrous

scaffolds. The first step is, obviously, the production of the polymeric fibers.

One of the methods available for obtaining fibers from oppositely charged

polyionic molecules is by interfacial polyelectrolyte complexation followed

by wrapping of the fibers in an appropriated roller. These fibers may then be

used to prepare the 3D matrices by, for example needle-punching. In this

case, needles punch through the layers of fibers, entangling them and turning

them into the final scaffold (119). This technique has so far been tested for

different polyelectrolytes complexes based on polysaccharides, namely for

combinations of chitosan/alginate (120), hyaluronate/chitosan, (121) and

chitosan/carboxymethylcellulose (122).

Another method for obtaining fibers from polyelectrolyte complexes is

by electrospinning. The main advantage of electrospinning is the possibility

of producing polymer fibers with diameters varying from 3 nm to larger than

5 µm (118). Another important factor is the simplicity of the setup applied,

which is usually composed of a syringe pump, a high voltage source, and a

collector (Fig. 8).

In this technique, a polymer solution is ejected through a charged

needle toward a grounded collector. As the process proceeds, the solvent

evaporates leaving the polymeric nanofibers deposited on the collector (123).

Figure 8. Representation of the typical setup for electrospinning technique.


Electrospinning has been used in the preparation of scaffolds based on

polyelectrolytes complexes also in different combinations, such as chitosan/

alginate (123), and chitosan/hyaluronic acid (121).

4.3.2. Sponges

As previously mentioned, scaffolds for tissue engineering must present a

porous morphology. Therefore, sponge-like materials, by fulfilling this

criterion, have been produced and applied as 3D matrices for tissue

regeneration. There are several forms of obtaining sponge-like structures

from polyionic molecules, and more specifically, for polysaccharides based

PECs. The simplest method is based on the mixture of at least two oppositely

charged polyelectrolytes. If parameters like molecular weight and

stoichiometry are controlled, phase separation may occur and an aggregate

(PEC) is obtained. The formed PEC may then be freeze-dried and after water

removal, a highly porous material is obtained (Fig. 9).

Pectin/chitosan sponge-like scaffolds have been prepared by the freeze

drying of the insoluble PECs formed by these two polysaccharides in an

aqueous solution (124). In vitro studies with human osteoblast cells showed

that these adhered and proliferated on the surface of the scaffold, proving the

biocompatibility and non-citotoxicity of the biomaterial. A similar method

was applied by the same group to produce a porous biodegradable matrix to

be applied as a scaffold for dental pulp regeneration (125). In this case, a

mixture of hyaluronan and chitosan was used in PECs preparation. The

biocompatibility of the obtained sponges was accessed in vitro using

mesenchymal stem cells and the materials proved to be biocompatible.

Figure 9. Scanning electronic microscopy image of a sponge based on a pectin/chitosan

PEC.


4.3.3. PEMs films

Polyelectrolyte multilayer films are prepared by the layer-by-layer

method previously described. This method has several advantages such as the

fact of not requiring expensive equipment and also the ability that PEMs

present in preserving the activity of biological molecules (126). PEMs

assembly has been widely applied to several polysaccharides like chitosan

(127), hyaluronic acid (128), alginate (129) and dextran sulfate (130).

Various studies have so far been reported concerning their application in

tissue engineering.

In a very ambitious yet successful experiment, Kim and Rajagopalan

(131) designed an in vitro hepatic model in which two layers of hepatic cells,

hepatocytes and liver sinusoidal endothelial cells (LSECs) were separated by

an intermediate chitosan-hyaluronic acid polyelectrolyte multilayer scaffold

(Fig. 10). This PEM scaffold function was to mimic the Space of Disse, a

protein-enriched, charged interface that separates hepatocytes and LSECs in

the liver. These 3D liver models maintained the phenotype of both cell types

simultaneously for up to 4 weeks.

A lot remains unsaid about the application of polyelectrolyte complexes

in tissue engineering. In fact, every year the number of published papers in

this area of research is impressive. The purpose of replacing human tissues

and organs without constrains of donor’s shortage and immunogenicity

problems will most certainly keep the interest of researchers and investors in

this area of knowledge.

Figure 10. 3D liver model developed by Kim and Rajagopalan (131) composed of

hepatocytes and LSECs separated by an intermediate chitosan-hyaluronic acid

polyelectrolyte multilayer scaffold.


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