Chitosan nanocapsules Effect of chitosan molecular weight and acetylation degree on electrokinetic

Colloids and Surfaces B:Biointerfaces 82 (2011) 571–580

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Colloids and Surfaces B:

Biointerfaces

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r f

b

Chitosan nanocapsules:Effect of chitosan molecular weight and acetylation degree on electrokinetic behaviour and colloidal stability

M.J.Santander-Ortega a ,b ,J.M.Peula-García c ,F.M.Goycoolea b ,J.L.Ortega-Vinuesa d ,?

a

Department of Pharmaceutical and Biological Chemistry,The School of Pharmacy,University of London,London WC1N 1AX,UK

b

Department of Pharmacy and Pharmaceutical Technology,University of Santiago de Compostela,Santiago de Compostela 15706,Spain c

Department of Applied Physics II,University of Malaga,Malaga 29071,Spain d

Department of Applied Physics,University of Granada,Granada 18071,Spain

a r t i c l e i n f o Article history:

Received 2July 2010Received in revised form 21September 2010

Accepted 11October 2010

Available online 15 October 2010Keywords:

Lipid nanocapsules Chitosan shell Colloidal stability Hydration forces

Chitosan hydrophobicity

a b s t r a c t

In recent years,chitosan nanocapsules have shown promising results as carriers for oral drug or peptide delivery.The success in their applicability strongly depends on the stability of these colloidal systems passing through the digestive tract.In gastric ?uids,clear stability comes from the high surface charge density of the chitosan shell,which is completely charged at acidic pH values.However,in the intesti-nal ?uid (where the pH is almost neutral)the effective charge of these nanocapsules approaches zero,and the electrostatic forces cannot provide any stabilization.Despite the lack of surface charge,chi-tosan nanocapsules remain stable in simulated intestinal ?uids.Recently,we have demonstrated that this anomalous stability (at zero charge)is owed to short-range repulsive forces that appear between hydrophilic particles when immersed in saline media.The present work examines the in?uence of the chitosan hydrophobicity,as well as molecular weight,in the stability of different chitosan nanocapsules.A study has been made of the size,polydispersity,electrophoretic mobility,and colloidal stability of eight core–shell nanocapsule systems,in which the chitosan-shell properties have been modi?ed using low-molecular-weight (LMW)and high-molecular-weight (HMW)chitosan chains having different degrees of acetylation (DA).With regard to the stability mediated by repulsive hydration forces,the LMW chitosan provided the best results.In addition,contrary to initial expectations,greater stability (also mediated by hydration forces)was found in the samples formed with chitosan chains of high DA values (i.e.with less hydrophilic chitosan).Finally,a theoretical treatment was also tested to quantify the hydrophilicity of the chitosan shells.

? 2010 Elsevier B.V. All rights reserved.

1.Introduction

During 1950s,some biopharmaceutical and pharmacokinetic studies related to the controlled release of drugs were developed,spurring scienti?c interest in this topic [1].Since then,the devel-opment of nanoparticles as drug delivery systems has considerably improved.The huge number of preparation methods and raw mate-rials employed in developing new drug delivery systems accounts for their enormous relevancy [2].Among these,lipid-colloidal systems based on a core–shell structure formed by an oily core surrounded by a polymeric shell have offered promising results for the vehiculization of several hydrophobic bioactive molecules [3].These reservoir formulations present different advantages,such as high drug encapsulation ef?ciency,low polymer content and reduc-tion of the tissue irritation due to the polymeric shell [4,5].The speci?c properties of the polymer that forms the shell will deter-

?Corresponding author.Tel.:+34958240018;fax:+34958243214.E-mail address:jlortega@ugr.es (J.L.Ortega-Vinuesa).mine the biodistribution,selectivity and the long-term stability of the nanostructure [6].Chitosan has recently been proposed as an ideal component of the polymeric shell of oily nanocapsules due to its advantageous biological properties,such as biodegradability,biocompatibility,mucoadhesivity,and permeability enhancement [6–11].In addition,chitosan-coated nanocapsules formulated by a solvent-displacement technique [12]have demonstrated the ef?-cacy of this polymer in the vehiculization of drugs such as capsaicin [13],calcitonin [14],and docetaxel [15].Unfortunately,despite these advantages,the p K a value of the glucosamine groups of chitosan usually goes from 6to 7,depending on its degree of acety-lation (DA)and molecular weight (MW)[13].Consequently,the super?cial net charge of chitosan nanocapsules would be close to zero in most biological ?uids,including parenteral or intestinal media,which could lead to the aggregation of these systems,and thus render them useless.

Nevertheless,previous studies conducted in our laboratory [16]have demonstrated that chitosan located in the shell of these nanocapsules is able to improve their colloidal stability,even at the isoelectric point of the particles (pH 7),provided that the medium

0927-7765/$–see front matter ? 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2010.10.019

572M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces82 (2011) 571–580

in which they are immersed has moderate or high salinity.This anomalous stabilization,explained neither by the classical DLVO theory nor by the steric repulsions among polymer chains,is given by short-range repulsive interactions known as hydration forces [13,16,17].Although the stability of many colloidal systems can be understood by considering the well-known DLVO theory[17],the theory also presents some serious limitations,as even stated by one of its authors[18].Water near any hydrophilic surface cannot be considered a continuous medium.In hydrophilic interfaces,water molecules and hydrated counterions are structured in the proxim-ity of the particle surface.These local ion-solvent structures close to the hydrophilic interface block the collision of two approach-ing colloidal particles,since these ordered“ion-water”layers must be removed to permit intimate contact between the two particle surfaces.However,the removal of these layers from hydrophilic surfaces is energetically unfavourable,and this prompts structural repulsive interactions known as hydration forces.Because of the hydrophilicity of the chitosan shell[19],hydration forces must be taken into account for the proper analysis of the colloidal stability of lipid nanocapsules coated by this polysaccharide.

Most of the properties of any colloidal system depend on the surface characteristics of its particles.Therefore,in nanocapsules coated by chitosan,the effective surface charge,electrokinetic mobility,and colloidal stability will depend exclusively on the physicochemical properties(DA and MW)of the chitosan chains immobilized at the interface.It is known that polymers based on the chitosan chemistry display different conformations.For low DA values(<20%)chitosan presents a hydrophilic polyelec-trolyte behaviour and the intramolecular electrostatic repulsions will favour the formation of an open,stiff structure.At a higher DA,the nature of the backbone becomes partially hydrophobic and,thus,hydrophilic and hydrophobic interactions are counter-balanced.Under these conditions the intramolecular repulsions decrease and the conformation of the polymer becomes more?ex-ible.For DA>50%hydrophobic interactions predominate,creating attractive intramolecular interactions which promote the forma-tion of loop-like assemblies[20].This behaviour is also affected by the MW of the polymer[21].All these phase transitions affect not only the structure of chitosan in solution but also to its confor-mation when it is adsorbed onto an oily core[22].If we take this situation into account,we might be able to modify the arrange-ment of the hydrophilic/phobic moieties of chitosan polymer onto the oily core by selecting the speci?c MW and DA of chitosan and thereby modulate the hydrophilic character of the nanocapsules.

In this scenario,different nanocapsules formulated with chi-tosan of low(11kDa)and high(122kDa)molecular weight (denoted as LMW and HMW,respectively)and four different DA (from1.4%up to56%)were prepared by a solvent-displacement technique[23].Their electrokinetic behaviour and colloidal sta-bility in the presence of different salts,as well as in commercial biological media,were characterized.As will be shown in this paper,the stability of the particles under physiological conditions depended entirely on the characteristics of the chitosan,and we demonstrate that hydration forces played a crucial role in keep-ing these particles stable in solutions for which the DLVO theory predicts aggregation.

2.Materials and methods

2.1.Reagents

A series of chitosan samples were prepared and puri?ed from a parent batch of chitosan obtained from chitin derived from squid pen supplied by Dr.Dominique Gillet of Mahtani Chitosan Pvt. Ltd.(France).For this,puri?ed chitosan was depolymerized under nitrous acid generated from NaNO2[24]in order to obtain two chi-tosan batches,i.e.of a high and low degree of polymerization(MW ～122kDa and11kDa,respectively,as determined by HPLC SEC-MALLS),hereafter referred to as HMW chitosan and LMW chitosan, respectively.Different aliquots of HMW and LMW chitosan sam-ples were further N-acetylated under homogeneous conditions by adding the needed stoichiometric amount of acetic anhydride in 1,2-propanediol[21]so as to afford chitosan with different degrees of acetylation(DA)–determined by1H NMR–varying in the range 1.6–56%for the HMW chitosan,and1.4–51%for the LMW sample. Lecithin(Epikuron145V)was from Degussa(Spain);Miglyol812?(caprylic/capric triglycerides)was supplied by Lemmel(Spain).In order to study the electrophoretic mobility and colloidal stability at different pH values,several buffered solutions with a low ionic strength(I=0.002M)were prepared:pH4.0and5.0were buffered with acetate,pH6.0and7.0with phosphate,and pH8.0and9.0 with borate.

2.2.Nanocapsule preparation

Chitosan-based nanocapsules were prepared following the pro-tocol originally developed in our laboratory[23],slightly modi?ed by avoiding the use of any poloxamer in the aqueous phase. Brie?y,20mg of lecithin were dissolved into250?L of ethanol, then62.5?L of Miglyol812?were added,followed by addition of4.75mL of acetone.Immediately afterwards,this organic phase was poured into10mL of an aqueous solution containing chitosan (0.5mg/mL)in5%stoichiometric excess of acetic acid.Immediately upon addition of the organic phase,the chitosan solution turned milky.Acetone,ethanol and a portion of the volume of water were evaporated in a rotavapor at40?C for～8min,giving a?nal volume corresponding to one-third of the original one.As a reference,an uncoated nanoemulsion(NE)was prepared under an identical pro-tocol as that used for chitosan-coated nanocapsules,but without including chitosan in the aqueous phase.

2.3.Size and storage stability

The average size of the nanocapsules was determined by photon-correlation spectroscopy(PCS)with a commercial light-scattering set-up,4700C,Malvern Instruments(Malvern,UK),with an argon laser of wavelength 0=488nm,working at a?xed angle (90?)at25?C.PCS gives information about the average diffusion coef?cient of the particles,which can be easily related to the mean diameter(?)using the Stokes–Einstein equation for spheres.The average size of our samples–which was measured weekly–was constant for months,this being an indication of high stability,at least when kept in the storage medium(puri?ed water,4?C).

2.4.Electrophoretic mobility

A ZetaPALS instrument(Brookhaven,USA)was used to mea-sure the electrophoretic mobility( e).The study was focused on measuring the e as a function of pH while maintaining a constant low-ionic-strength value(0.002M).Each e mobility datum was the average of45individual measurements.

2.5.Colloidal stability

NaCl,CaCl2,and MgCl2were used as destabilizing agents. According to the classical DLVO theory[17],increased salinity triggers the coagulation of a lyophobic colloidal system.During aggregation,the turbidity of the system increases when the average size of the scattering particles enlarges.Therefore,a simple spec-trophotometer(Bio-rad680,microplate reader)working with a visible wavelength( =570nm)is clearly able to detect and analyse

M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces82 (2011) 571–580573

Table1

Chitosan degree of acetylation(DA),hydrodynamic mean size of the particles,polydispersity index(PDI), -potential at pH6( ),Stern potential(?),and hydration constant (C h)of our nanocapsules.The last two parameters were estimated by?tting the theoretical interaction energy given by Eq.(3)to the ccc and csc values shown in Fig.4(only for NaCl),as explained in Theoretical appendix.

Chitosan MW(kDa)DA(%)Size(nm)PDI (mV)?(mV)C h×1020(J)

LMW11 1.4150±30.1243.68.6512.1

9.0160±20.1437.48.5013.0

27.0148±10.1032.67.7022.7

51.0159±10.1128.7 6.0031.9

HMW122 1.6183±70.0856.312.20 1.7

11.0185±30.0648.611.25 3.2

27.0177±60.1144.211.10 3.6

56.0183±50.1141.810.007.4

the aggregation https://www.360docs.net/doc/4d17492021.html,rmation on the kinetics-aggregation

constant“k”of dimmer formation can be derived from the initial

slopes of the absorbance vs.time curves(dAbs/dt)by[25–27]:

dAbs dt =

C2/2?C1

N2

l

2.3

k(1)

where C1and C2are the scattering cross-sections of a monomer and a dimmer,respectively,N0is the initial particle concentration, and l is the optical path through the cuvette.Nevertheless,stability is usually evaluated by calculating the Fuchs factor(W),instead of calculating the k values using Eq.(1).The Fuchs factor(also called “stability factor”)is related to the number of collisions that two colliding particles must undergo before they remain de?nitively stuck.Therefore,when W=1the system is completely unstable, while W=∞indicates total stability.It is easy to calculate the Fuchs factor at each salt concentration from the aggregation curves using the following equation:

W=k f

k s

=(dAbs/dt)f

(dAbs/dt)s

(2)

where“k f”refers to the fastest aggregation-kinetics constant,and the subscript“s”refers to slower coagulation rates.Plotting W as a function of the medium salinity in a double-logarithmic scale becomes useful to estimate the critical coagulation concentration (ccc)–the point where W reduces to1–and the critical stabi-lization concentration(csc)–the point where W increases from 1to∞when salt concentration increases even more–which are fundamental parameters in colloidal stability studies.More details concerning the way in which W,ccc and csc values can be derived experimentally can be found in Refs.[16,19].The ccc value is related to destabilization processes and indirectly gives information on the surface-charge density of the particles;thus,a low ccc means low stability.However,the csc value–de?ned as the minimum salt con-centration at which the system begins to re-stabilize when salinity is increased even more–is associated with surface hydrophilicity. It should be noted that csc values increase when the hydrophilic-ity decreases,since the higher the hydrophilicity of the surface the lower the concentration of hydrated ions needed to create a struc-tural barrier,and viceversa(the lower the hydrophilicity,the higher the csc).Therefore,a low csc value means high hydrophilicity.

2.6.Stability in biological media

The colloidal stability of the nanocapsules formulated was investigated in the following cell-culture media:(a)Opti-MEM 1×(added with GlutaMAX-1,GIBCO)at pH7.45;(b)RPMI-1640 medium(PAA Laboratories GmbH)at pH7.34without any supple-ment;(c)RPMI-1640medium supplemented with6%(v/v)fetal bovine serum(PAA Laboratories GmbH),1%(v/v)l-glutamine and 0.2%(v/v)penicillin/streptomycin solution(GPS,Sigma–Aldrich)at pH7.34;(d)simulated gastric(pH1.2),and(e)intestinal(pH6.8)?uids,both prepared according to the United States Pharmacopeia XIX.Finally,the colloidal stability was also analysed in more simple biological media such as a(f)phosphate-buffered saline solution (PBS,Sigma–Aldrich)and a(g)D-PBS solution(Sigma–Aldrich), both at pH7.4.

3.Results and discussion

Size is a critical parameter that controls the potential use of these colloids as successful drug delivery systems.It has been demonstrated that the optimum mean diameter to cross biolog-ical barriers is around or just below of200nm[28–30].In this sense,all the particles used were potentially apt for delivery sys-tems,since their diameters were in the150–185nm range(see Table1).The nanocapsule size was dependent on the molecular weight of the chitosan,although it was not affected by the DA at all.Chitosan,coating the particle,adheres to the lecithin layer by means of attractive electrical forces(in the low DA cases)[31] together with hydrophobic interactions(only in the high DA cases) [32,33].Therefore,the differences observed in size appear to arise from the different thickness of the shell,this being much thicker in the HMW samples.The amount of chitosan immobilized in the nanocapsules was calculated following Muzzarelli[34].It should be noted that the incorporation of chitosan was consistently higher than90%,being almost complete(99%)for the highest DA cases (LMW51%and HMW56%)[13].This clearly indicates that the attractive hydrophobic interactions between the hydrophobic frag-ments of the acetylated chitosan and the lipid core or hydrophobic patches of the lecithin layer favour adsorption,complementing the electrostatic attraction among the charged parts of the lecithin and chitosan.

The electrical state of the particle surface is other physical parameter that needs to be evaluated.This information can be gained by electrophoretic mobility( e)measurements.Fig.1 shows the e data of the LMW samples as a function of pH.Similar results were found with the HMW nanocapsules.The incorpora-tion of chitosan on the surface of the nanocapsules was clearly manifested in these experiments,once the e data of nanocap-sules coated by chitosan were compared with those found with an uncoated nanoemulsion.Differences were much more evident under acidic conditions;at acid pHs the glucosamine groups of chi-tosan located in the shell converted negative particles into positive ones.As can be seen,the acetylation degree affected the mobility data,since the higher the acetylation degree,the lower the num-ber of positive charges on the surface(that coexist with a?xed amount of negative charges supplied by the lecithin layer).In this sense,the isoelectric point(iep)of the particles–i.e.the pH at which e reduces to zero–decreased when the DA was increased. The approximate iep values given by Fig.1were7.8,7.6,7.3,and 7.1,for DA values of1.4%,9%,27%,and51%,respectively.A simi-lar tendency was observed with the HMW systems.The reduction in charged glucosamine groups in the chitosan shell caused by the acetylation process is also re?ected in Fig.2,where the e data are

574M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces 82 (2011) 571–580

2

4

μe 108 (m 2

/ V s )

pH

Fig.1.Electrophoretic mobility of our LMW samples as a function of

pH.The degree of acetylation of the chitosan shell was:1.4%( ),9%(),27%( ),and 51%().The mobility of a nanoemulsion prepared without including chitosan in its synthesis was also measured ().

60

504030201002,5

3,0

3,5

4,0

4,5

5,0

5,5

μe 108

(m 2

/ V s )

DA (%)

Fig.2.Electrophoretic mobility at pH 4.0vs.the degree of acetylation.LMW ()

and HMW ()samples.

plotted as a function of DA.It should be noted that,in general,the mobility of the HMW samples was higher than that of the LMW ones,indicating (as expected)that the effective surface charge in a thicker chitosan layer (HMW)was higher than in thin chitosan shells (LMW).Finally,an additional study was made to evaluate whether DA alters the average p K a of the particles.The average p K a values of the surface groups were estimated,taking into account the data shown in Fig.1,by ?tting the sigmoidal e curve to an equa-tion given in Ref.[35].The estimated p K a values were coincident in all cases,?nding an average value of 7.0±0.2.

The next set of experiments was performed to evaluate the col-loidal stability of the particles at pH 6.0in presence of different electrolytes (NaCl,CaCl 2,and MgCl 2).These experiments were use-ful not only to estimate the effective surface charge (by means of the ccc values),but also to gain information about the hydrophilic-ity of the outer part of our particles through the csc values.Fig.3a and b shows a typical stability experiment for the HMW samples with NaCl and the LMW samples with CaCl 2,respectively.The ccc and csc values thus calculated were used to generate a stability diagram (see Fig.4).The shady areas are delimited by the ccc and csc values,and they represent salinity conditions at which the par-ticles rapidly aggregate.The csc values delimit the upper part of

W

[NaCl] (mM)

10

1

110

100

W

[CaCl 2] (mM)

Fig.3.(a)Stability factor of our HMW nanocapsules as a function of NaCl concen-tration.1.6%( ),11%(),27%( ),and 56%().The straight solid lines help to guide

the eye toward the ccc values,while the dash lines serves to locate the csc values.(b)Stability factor of our LMW nanocapsules as a function of CaCl 2concentration.1.4%( ),9%(),27%( ),and 51%().The straight solid lines help to guide the eye toward the ccc values,while the dash lines serves to locate the csc values.

these instability regions,while the lower part is de?ned by the ccc data.Once the salinity value exceeds the csc values,the repul-sive hydration forces begin to act,and the stability improves when the salt concentration increases even more.Likewise,below the ccc ,there is a potential barrier that hinders the collision of two approaching particles.According to the DLVO theory,the lower the salt concentration,the higher the height of this barrier,and in turn the stronger the stability of the system.If this is true,any system that is unstable under physiological salinity would become use-less for future applications as drug carriers,since it would rapidly aggregate.

There are some general ?ndings that deserve to be discussed:(1)The trend of the ccc values agrees with the DA values (and with

the electrophoretic mobility behaviour):the higher the acety-lation degree the lower the number of charged glucosamine groups,yielding to less stable particles.This trend appears in both systems (LMW and HMW)regardless of the salt used.(2)Once a given salt is chosen (NaCl,CaCl 2,or MgCl 2),the ccc

values of the HMW samples become higher than those found with the LMW particles.This result also agrees with the surface charge density of the chitosan shell.Chitosan chains are posi-tively charged at pH 6.0,and thus,the thicker the chitosan layer

M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces82 (2011) 571–580

575

Fig.4.Stability diagrams of our nanocapsules at pH6.0for three different elec-trolytes(NaCl,CaCl2,and MgCl2).The dark areas represent salt concentrations at which instability is complete,as they correspond to regions above the ccc but below the csc.The horizontal black line represents the usual ionic strength value found in most of the biological?uids.If this line crosses the instability region of a given system,this means that such a system would be useless for practical applications, since it would rapidly aggregate.

(HMW cases),the higher the charge density and the higher the stability according to the DLVO theory.

(3)Saline-mediated repulsive hydration forces arise in the eight

systems studied,this being a clear indication of the effective incorporation of chitosan–a hydrophilic polysaccharide–onto the particle surface.However,a striking result was found when the hydrophilicity of the chitosan shell was analysed.The csc values consistently decreased(i.e.the average hydrophilicity of the surface increased)with the DA,when the glucosamine groups were partially changed by hydrophobic acetylated frag-ments.This unexpected result constitutes a general trend found independently with each electrolyte.A plausible explanation for this?nding will be given below.

(4)If hydrophilicity is analysed with regard to the molecular

weight of the chitosan,the LMW nanocapsules show a more hydrophilic character(lower csc values)than the HMW parti-cles.

(5)Finally,taking into account the csc values again,we found the

stability provided by the hydration forces to be much stronger (lower csc values)when divalent cations were present in the medium.It should be noted that the magnitude of these repul-sive structural forces is not only correlated to the hydrophilicity of the surface,but also strongly depends on the concentra-tion and hydration degree of the ions surrounding the surface [32,36–39].For this reason,Ca2+or Mg2+always exert higher restabilization effects–lower salt concentrations are neces-sary to generate repulsions mediated by hydration forces–than sodium,as these divalent cations are much more hydrated than Na+[32].Although hydration repulsions have usually been ascribed to the role played by counterions(Cl?in our experi-ments working at pH6.0),it has been demonstrated that coions also participate in these kinds of repulsive interactions[40].It might be worthy to comment that,although it is well-known that divalent cations speci?cally interacts with the lecithin polar groups[41,42],it is more than likely that such interac-tions do not take place in our systems,since the lecithin charges are hidden(and counterbalanced)by the positive charges of the chitosan layer that coats the particle.This protective shell

avoids direct interaction between calcium or magnesium with the lecithin layer.

It is worth discussing the points3and4above in more detail.The reason why the average hydrophilicity of the outer part of our nanocapsules increases with the chitosan DA must be sought in the hydrophobic interactions that take place between the hydrophobic moieties of the chitosan(with high DA)and the hydrophobic patches of the lecithin layer and/or the oily core. The term hydrophobic interaction refers to the spontaneous dehy-dration and subsequent approximation and contact of non-polar components in an aqueous environment.The dehydration origi-nates from the fact that interactions between water molecules are far more favourable than contacts between non-polar groups or between a non-polar group and water.Hence,non-polar groups tend to be rejected from an aqueous environment rather than being attracted to one another.Hydrophobic interaction is characterized by a large entropy increase(given by the release of structured water molecules that were in contact with non-polar groups)and a relatively small enthalpy effect.Taking this situation into con-sideration,we found that when a chitosan chain has a very low degree of acetylation(i.e.1.4%),the polysaccharide backbone is very hydrophilic.Therefore,hydrophobic interactions cannot take place between this chitosan and the lecithin/oil nanocapsule,and attractive interactions among the glucosamine groups(of the chi-tosan)and the negatively charge groups of lecithin are purely electrostatic[31].This means that,if any hydrophobic patch is pre-sented in the oil/lecithin nanoemulsion,this will be not coated by the electrostatically deposition of chitosan,and subsequently, the?nal nanocapsule formed with low DA chitosan will exhibit the original hydrophobic patches.However,when chitosan with a relatively high DA value is used instead(i.e.DA～50%),the inter-action between this polysaccharide and the lecithin/oil surface is not only electrostatic,but also is driven by hydrophobic forces.The hydrophobic interaction will take place between the hydropho-bic moieties of the chitosan chain and the hydrophobic parts of the lecithin/oil surface,both of them avoiding contact with water and orienting the hydrophilic parts toward the aqueous phase.As a result,the hydrophilicity of the outer surface of our high-DA chi-tosan/lecithin/oil nanocapsules is stronger than that exhibited by low-DA chitosan,since the original hydrophobic patches in the particles are now hidden.This would explain why more hydropho-bic chitosan(high DA)yields to more hydrophilic surfaces in our nanocapsules,that is,lower csc.

To quantify the hydrophilicity of the nanocapsule surface,we carried out a theoretical treatment of our experimental results.This treatment is detailed in Appendix of this paper.By?tting the exper-imental stability data(ccc and csc values)to theoretical equations in which DLVO and hydration forces are considered,we?nd it pos-sible to evaluate the hydrophilic character of a surface.Quantitative information concerning the hydrophilicity is given by the param-eter“hydration constant”(C h),which depends only on the surface nature,but not on the ions dissolved in the medium.A high C h value means high hydrophilicity.Table1shows the C h values found for our systems.Two conclusions can be drawn:(i)the hydrophilic-ity of the nanocapsule surface in fact increases with the chitosan DA,as explained above,and(ii)those particles synthesised with LMW chitosan exhibit a much more hydrophilic surface than do those obtained with HMW chitosan.The reason for this is because short polymeric chains(LMW)can re-accommodate their spatial conformation by hiding their hydrophobic areas from water con-tact much more easily than can a long chain(HMW).However,by increasing its molecular weight,chitosan will form loop-like struc-tures to protect its hydrophobic moieties from the aqueous phase [20,21].These intramolecular interactions make dif?cult the above commented re-structuring of the polymer onto the oily core,and

576M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces 82 (2011) 571–580

1

1/2

1/3

1/5

1/10

1/15

1/20

1/30

0,000

0,0010,0020,0030,0040,005

0,006

d (A b s )/d t (s -1

)

Dilution with regard to the original medium 1

1/2

1/3

1/5

1/10

1/15

1/20

1/30

0,000

0,001

0,002

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d (A b s )/d t (s -1

)

Dilution with regard to the original medium

1

1/2

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1/5

1/10

1/15

1/20

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0,000

0,0010,0020,0030,0040,005

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d (A b s )/d t (s -1

)

Dilution with regard to the original medium 1

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1/15

1/20

1/30

0,000

0,001

0,002

0,003

0,004

0,005

0,006

d (A b s )/d t (s -1

)

Dilution with regard to the original medium

Fig.5.Aggregation kinetics of (a)LMW with a DA =1.4%,(b)LMW with a DA =51%,(c)HMW with a DA 1.6%,and (d)HMW with a DA =56%,vs.different dilutions of media with biological interest:( )PBS,( )D-PBS,( )Opti-MEM,(?)RPMI,and ( )RPMI supplemented with fetal bovine serum.

hence,the polymer probably is not able to completely protect some hydrophobic patches from exposure to the aqueous medium.

To evaluate the potential applicability of our particles as drug delivery systems in physiological environments,we made a new study of stability in different media of biological interest.For clarity we show only the results of our four limit cases:LMW with DA =1.4%(Fig.5a),LMW with DA =51%(Fig.5b),HMW with DA =1.6%(Fig.5c),and HMW with DA =56%(Fig.5d).In these ?g-ures,stability is correlated with the initial absorbance variation (dAbs/dt )of a solution in which the particles are added.When aggre-gation takes place,the turbidity of the solution increases,dAbs/dt being higher for the most unstable systems.Likewise,a dAbs/dt equal to zero means total stability.It should be noted that the media in which the experiments where conducted (described in Section 2)had moderate salinity,similar to that found in biolog-ical ?uids.Fig.5a–d shows a common feature in all our systems,irrespective the particle or the medium nature:all of these are sta-ble,in the sense that is,dAbs/dt =0in the original medium (point “1”in the x -axis).The origin of this stability may be hardly elec-trostatic,since all our media possess pH values very close to,or even coincident with,the isoelectric points of our nanocapsules.Although the electrical contribution to the stability in our biolog-ical media must not be completely neglected,it actually must be weak under these conditions,and consequently it will be ruled out in this discussion.However,hydration forces would explain the stability patterns observed.In fact,as soon as the salinity of the original medium was reduced by successive dilutions,all our stable systems began to aggregate,supporting the idea that the original stability was caused by the presence of a considerable amount of hydrated ions (i.e.reduced with dilutions).Above,it was explained that the magnitude of the repulsive hydration forces depends not only on the hydrophilicity of the surface,but also on the concentration of hydrated ions surrounding it.The higher the hydrophilicity,the lower the amount of salt needed to re-stabilize the system.Therefore,the results shown in Fig.5a–d also serve to order our systems according to their hydrophilicity.The most hydrophilic systems will be those in which a large dilution of the original salinity is necessary to appreciate aggregation.For exam-ple,samples with a high DA are much hydrophilic than those of low DA,since the former are stable even in media diluted 5-fold (see LMW DA =51%in Fig.5b)while aggregation starts at lower dilutions (2-or 3-fold)in low-DA samples (see LMW DA =1.4%in Fig.5a).A similar trend is found with the HMW samples (Fig.5c and d).Other systems with intermediate DA values (LMW with DA =9%or DA =27%,or HMW with DA =11%or DA =27%)showed a behaviour that was also intermediate between those shown in Fig.5.Conse-quently,these new experiments con?rm our previous conclusions:nanocapsules formed with chitosan with high a DA show a more hydrophilic surface than those synthesised with chitosan of a low DA.

M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces 82 (2011) 571–580577

1

1/2

1/3

1/5

1/10

1/15

1/20

1/30

0,000

0,0010,0020,0030,0040,005

a

b

c d

d (A b s )/d t (s -1

)

Dilutions with regard to the original medium

1

1/2

1/3

1/5

1/10

1/15

1/20

1/30

0,000

0,001

0,002

0,003

0,004

0,005

d (A b s )/d t (s -1

)

Dilutions with regard to the original medium

1

1/2

1/3

1/5

1/10

1/15

1/20

1/30

0,000

0,0010,0020,0030,0040,005

d (A b s )/d t (s -1

)

Dilutions with regard to the original medium 1

1/2

1/3

1/5

1/10

1/15

1/20

1/30

0,000

0,001

0,002

0,003

0,004

0,005

d (A b s )/d t (s -1

)

Dilutions with regard to the original medium

Fig.6.Aggregation kinetics in of (a)LMW with a DA =1.4%,(b)LMW with a DA =51%,(c)HMW with a DA 1.6%,and (d)HMW with a DA =56%,vs.different dilutions of simulated gastric ?uid (---),and simulated intestinal ?uid (—).

In addition,these experiments also demonstrate that particles with a shell made of low molecular chitosan are more hydrophilic than those with high MW (keeping the DA constant).For exam-ple,the LMW sample of high DA (Fig.5b)is stable even in media where the salinity is 5-fold lower than the original medium,while the HMW system with high DA (Fig.5d)becomes unstable from dilutions where salinity is a third of the original.A similar reason-ing can be applied to the systems with a low DA (Fig.5a and c).Therefore,the stability study performed in these biological media agrees with those carried out at pH 6and shown in Fig.4.

As chitosan nanocapsules are usually orally administrated,the last set of stability experiments was made in simulated digestive ?uids.Once again,only the four limit cases will be shown for the sake of clarity,although experiments were performed with our eight systems.Fig.6a–d shows the initial aggregation kinetics of nanocapsules immersed both in simulated gastric and in intestinal ?uids,as well as in successive dilutions.The stability was almost complete in gastric ?uid,since the chitosan shell was completely charged at pH 1.2.In this medium,stability is mainly provided by the electrostatic DLVO contribution.Only the nanocapsules with higher DA values,in which the charge density is lower due to the acetylation of glucosamine groups,showed a little tendency to aggregate,as dAbs/dt was not exactly zero in some dilutions (see Fig.6b and d).Nevertheless,these aggregation kinetics were almost negligible compared to those in the intestinal ?uid.The intestinal pH approximately matches the isoelectric points of our nanocap-sules.The lack of surface charge was responsible for the aggregation in those media in which the original salinity was reduced by dilu-

tion.However,some samples were stable in the original intestinal ?uid,where the ionic strength was around 200mM.This stability was ascribed to hydration repulsive forces,as they are capable of stabilizing an uncharged system when the ionic strength increases,provided that the surface is suf?ciently hydrophilic.Actually,there was no aggregation in the original ?uid in those samples with the highest DA value (LWM 51%,and HMW 56%),re?ecting higher hydrophilicity than in the samples with low DA:LWM 1.4%(which showed rather slow kinetics;Fig.6a)and HMW 1.6%(which showed signi?cant coagulation kinetics;Fig.6c).In addition,if the results are compared between samples of identical DA,but varying the molecular weight,these experiments demonstrate,once more,that low-molecular-weight chitosan gives more hydrophilic shells than do high-molecular-weight chains.For example,when the salinity of the intestinal ?uid is reduced to the half,the LMW 51%sample is almost stable (Fig.6b)while the HMW 56%rapidly aggregates (Fig.6d).Likewise,the LMW 1.4%system in the original intesti-nal ?uid is practically stable (Fig.6a),while the HMW 1.6%clearly coagulates.4.Conclusions

Different chitosan derivatives varying in molecular weight and degree of acetylation were successfully used to form nanocapsules with potential applications as drug delivery systems.Initially,the particle size and polydispersity of our eight systems were appro-priate for such a purpose.Not only the size,but also the surface properties depended on the nature of the chitosan located in the

578M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces82 (2011) 571–580 external shell of the colloidal particles.Some basic studies on elec-

trokinetic mobility as well as colloidal stability experiments have

enabled us to draw certain conclusions regarding both the electrical

state and the hydrophilicity of the nanocapsule surface,respec-

tively.The electrokinetic measurements indicated that the average

surface charge was reduced with DA,as the number of positively

charged glucosamine groups diminished proportionally with the

acetylation process.With regard to hydrophilicity,the hydration

forces were fundamental in making our nanocapsule systems sta-

ble in media of moderate or high salinity,even under conditions

where the surface charge was close to zero.A broad set of stabil-

ity experiments demonstrated that hydrophilicity increased with

the DA but decreased with the molecular weight of the chitosan.

Despite that an increment in DA raises the number of hydropho-

bic parts in the chitosan backbone,the attractive hydrophobic

interactions among these fragments and the hydrophobic areas

of the oil/lecithin particle expose the hydrophilic regions to the

aqueous phase,as well as the hydrophobic parts are hidden.There-

fore,the average hydrophilicity becomes stronger in the systems

formed by chitosan with high DA values than those with low DA.

Finally,the in?uence of the molecular weight of chitosan on the

hydrophilicity was also signi?cant,as low MW chains supply a more

hydrophilic shell.In this case,the hydrophobic fragments can be

re-accommodated much better on the surface–avoiding their con-

tact with water–than those parts in high-molecular-weight chains,

where it is more than likely that some hydrophobic moieties cannot

be hidden due to steric hindrances.

As colloidal stability is a key point to develop useful drug deliv-

ery systems,our results suggest that the most stable nanocapsules,

to be used in biological media with moderate salinity,are those

formulated with low-molecular-weight chitosan having a DA value

around50%.It should be noted that repulsive hydration forces are

the main factors responsible for the stability of chitosan nanocap-

sules in biological media.

Acknowledgments

The authors thank the?nancial support given by the projects

MAT2007-66662-C02-01(European FEDER support included,

MICINN,Spain),and P07-FQM2496and P07-FQM3099(Junta de

Andalucía,Spain).

Appendix A.Theoretical appendix

To explain the stability of hydrophilic colloids quantitatively,

Molina-Bolivar et al.developed an extension to the classical DLVO

theory including hydration forces and their dependence on the salt

concentration[43].Following an approximation proposed by Chu-

raev and Derjaguin[44]in which a“structural”term that accounted

the hydration interaction was added to the classical DLVO terms,

the net potential energy for the interaction between two colloidal

particles can be described by the algebraic sum of three potentials:

V T=V A+V E+V H(3)

where V A is the London–van der Waals dispersion energy,V E repre-

sents the repulsive interaction caused by overlapping the electrical

double layers of the particles,and V H is a term that accounts the

repulsive hydration interaction energy.

The van der Waals attraction takes the following extended form

[17]:

V A=?A

6

2a2

H(4a+H)

+2a

2

(2a+H)2

+ln H(4a+H)

(2a+H)2

(4)

where A is the Hamaker constant for particles interacting in a given medium(water in our case),a is the particle radius,and H is the distance between the surfaces of the two approaching particles.

According to the constant potential model and for symmetrical electrolytes,a reasonable expression to V E(for moderate potentials in the Stern layer,<50mV),is[45]:

V E=2 (a+ )εε0

4k B T

z i e

2

e??(H?2 )(5) where is the thickness of the Stern layer,εthe dielectric constant of the medium,ε0the vacuum permittivity,k B the Boltzman con-stant,T the absolute temperature,z i the valence of the ion,e the elementary charge,?is the Debye parameter,

?=

i

i e2z2

i

εε0k B T

1/2

(6) and is given by the following equation:

=tanh

z i e ?

4k B T

(7)

where?is the Stern potential.Actually,Eq.(7)is a good approxi-mation for?a 1and?<50mV.

With regard to the non-DLVO interaction(V H),it is assumed that the ion-water structures accumulated at the proximities of the par-ticle surface decay away from that interface exponentially.This is why,as a empirical rule,it depends exponentially on the interlayer thickness“H”[32].To account for the role played by the electrolyte concentration,Molina-Bolivar et al.[43]proposed the following equation where the pre-exponential term is directly proportional to salt concentration(c e):

V H= a(N A C h c e) 2e?H/ (8) N A is the Avogadro number,C h is a proportionality constant called“hydration constant”,its value depending on the surface hydrophilicity,and is the decay length.The value of depends on the hydration number of the hydrated counterion,and it is usually within the0.2–1.1nm range[43].

It should be noted that the pre-exponential factor depends on the salt concentration in such a way that under very low salin-ity conditions the V H term can be considered negligible against the DLVO terms(V A and V E).However,the repulsive hydrophilic term becomes more and more signi?cant in the total interaction energy(V T)as soon as the ionic strength of the medium increases. This effect has been depicted in Fig.7for the LMW DA=51%sys-tem.

The above equations are useful to gain quantitative informa-tion about the surface hydrophilicity of our nanocapsules once the C h parameter is estimated by comparing the theoretical predic-tions with the experimental ccc and csc values obtained with NaCl at pH6.0(data shown in Fig.4).Since chloride acts as counte-rion at this acidic pH,the value accounting for the size of the adsorbed ions of the Stern layer was taken from the literature:for Cl?, =0.33nm[32].For a given system immersed in media with salt concentrations below the ccc(where the V H contribution is not strong enough),the Stern potential?,included in the V E term,was used as a?tting parameter to match its experimental ccc.The ccc is de?ned by the minimum salt concentration in which the repulsive potential barrier vanishes(that is,when both V T and dV T/dH are equal to zero).In this process,the Hamaker constant“A”was set at a constant value also chosen from the literature:A=0.55×10?20J. The procedure described is shown in Fig.8for the HMW DA=56% sample,which had a ccc=37mM and a csc=100mM with NaCl(see Fig.4).With the?and A values thus determined we can move now to the non-DLVO regions(around the csc)where the hydra-tion forces become important.Eq.(8)has two?tting parameters, and C h,the former depending on the counterion hydration and

M.J.Santander-Ortega et al./Colloids and Surfaces B:Biointerfaces 82 (2011) 571–580

579

-50

050100150200

250300350V /k B T

H (nm)

Fig.7.Effect caused by the hydration interaction (V H )when it is included in the DLVO interaction (V T =V A +V E )for the LMW DA =51%system in a solution containing a NaCl concentration of 150mM (which is higher than the corresponding ccc and csc values).The following parameters were set at:A =0.55×10?20J, ?=6.0mV, =0.33nm, Cl ?=0.35nm,and C h =31.9×10?20J.

-5

5

10

15

V T /k B T

H (nm)

Fig.8.Variation of the total interaction potential vs.distance when the NaCl con-centration was increased in the HMW DA =56%system.Both V T and dV T /dH become zero at the ccc and csc values The following parameters were set at:A =0.55×10?20J, ?=10.0mV, =0.33nm, Cl ?=0.35nm,and C h =7.35×10?20J.

the latter depending exclusively on the surface hydrophilicity of the particles.Nevertheless,the value for chloride was calculated in Ref.[16],being equal to 0.35nm,and thus,only C h remains as a ?tting parameter.In fact,this hydration constant was adjusted to match the experimental csc ,in which both V T and dV T /dH were also equal to zero (see Fig.8).This laborious procedure enabled us to estimate the ?and C h values for our eight systems when immersed in NaCl solutions at pH 6.0(Fig.4).Data are shown in Table 1.The Stern potential values agree with the electrokinetic mobility results (which are dependent on the -potential),and they decrease when the acetylation degree increases,as expected.In addition,this potential is higher when the shell was formed by chitosan of high molecular weight,as the charge density in this thicker layer is higher than in the LMW cases.It would be infor-mative to compare the theoretically calculated ?values with the -potential ones.Our electrophoretic instrumental device auto-matically transformed the mobility data into -potential values by means of the Helmholtz–Smoluchowski equation [46].The so obtained -potential results obtained at pH 6are also given in

Table 1.Both electrical potentials ( ?and )have similar trends,but they differ each other quantitatively.This quantitative,but not qualitative,discrepancy might be caused by the method used to transform mobility into -potential data.With regard to the surface hydrophilicity,C h gives quantitative information previously com-mented in the main text:that is,low-molecular-weight chitosan produces shells that are more hydrophilic than those formed by HMW chains.In addition,for a given molecular weight,an increase in the DA value yields to an increase in the average hydrophilicity of the surface.References

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