The zeta potential and surface properties of asphaltenes obtained with different crude oil/n-heptane proportions☆

Chitosan -Cholesterol and Chitosan -Stearic Acid Interactionsat the Air -Water Interface †

Hilda Parra-Barraza,‡ Marıa G. Burboa,‡,§ Mario Sanchez-Vazquez,‡ Josue Juarez,‡

Francisco M. Goycoolea,| and Miguel A. Valdez*,‡,⊥

Departamento de Investigacion en Materiales, Departamento de Investigaciones Cientıficas yTecnologicas, and Departamento de Fısica, Universidad de Sonora, Rosales y transversal,

C. P. 83000 Hermosillo, Sonora, Mexico, and Laboratory of Biopolymers, Centro de Investigacion enAlimentacion y Desarrollo, P.O. Box 1735, Hermosillo, Sonora 83000, Mexico

Received February 9, 2005; Revised Manuscript Received March 31, 2005

We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified bydifferent chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weightchitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol-chitosanand stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulationwas performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isothermsas a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. Thesemodifications were dependent on the type and concentration of chitosan. Severe modifications of all phaseswere noticed with larger molecular areas, and the observed changes in the compressional modulus weredependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible thanthe ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by thecholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. Thehydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weightchitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting withcholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds betweenthese molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showedthat the medium and high molecular weight chitosans could retain higher proportions of cholesterol comparedwith the other analyzed samples.

Introduction

Chitosan is a positively charged polysaccharide conven-tionally prepared by alkaline deacetylation of chitin,1,2 whichis extracted from the skeletal structure of crustaceans, insects,mushrooms, and the cells of fungi. Chitosan has a potentialuse in several areas, including application in cosmetics,3

biotechnology, and medicine.4 In these applications, chitosaninteracts in most cases with biomembrane surfaces. It hasbeen suggested that chitosan can be used as a hypocholes-terolemic agent when supplied to the diet.5 Its cationic natureleads to a strong interaction with lipids having an oppositecharge. Although the mechanism of this interaction is notwell understood, it was suggested, as one of the possiblecauses, the formation a complex between polyelectrolytesand lipid droplets in the intestine. The interaction between

chitosan and emulsified lipids has been analyzed by Fa¨ldtet al.6 They suggest that chitosan covers negatively chargedlipid particles in the intestine and this process is likely toinfluence the resorption of lipids in the intestinal tract.

Chitosan is being considered in various countries as adietary supplement of human consumption. Nevertheless,concern has arisen about the fact that certain promotionalcampaigns advertise chitosan as a fat-binding active agenttoward all kinds of lipids, with no supporting scientificevidence.7 Information about the interaction of chitosan withlipids is scarce. The chitosan and lecithin interactions werestudied by Magdassi et al.,8 and Sathirakul9 compared thelipid binding capacity of chitosan in terms of the oil holdingcapacity, cholesterol binding capacity, and bile acid capacity.As Muzzarelli et al.7 have mentioned, it is evident that thereis interest in examining the lipid collection ability of highlycharged chitosans (i.e., of a very low degree of acetylation,DA) and hydrophobically modified chitosans to verifywhether these characteristics are key to this phenomenon.On the other hand, the introduction of hydrophobic moleculargroups has been indicated as a cause of lipid sequesteringby chitosans or by chitosan-bile acid associations. Theyconclude that certain olive oil components may preferentiallybe retained in the oil fraction that imbibes the chitin or the

* To whom correspondence should be addressed. Miguel Angel Valdez,Depto. de Fı´sica, Universidad de Sonora, Rosales y Transversal, 83 000Hermosillo, Sonora, Me´xico. E-mail: [email protected].

† This paper was presented at the III Iberoamerican Symposium on Chitin(III SIAQ), held in Crdoba, Spain, September 27-29.

‡ Departamento de Investigacio´n en Materiales, Universidad de Sonora.§ Departamento de Investigaciones Cientı´ficas y Tecnolo´gicas, Univer-

sidad de Sonora.| Centro de Investigacio´n en Alimentacio´n y Desarrollo.⊥ Departamento de Fı´sica, Universidad de Sonora.

2416 Biomacromolecules 2005,6, 2416-2426

10.1021/bm050106s CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 05/06/2005

chitin derivative. They pointed out that certain steroidsinteracted effectively with polysaccharides and this wouldimply that cholesterol might behave analogously.

The interaction of lipids with some macromolecules hasbeen investigated at the air-water interface with the helpof the Langmuir balance, taking into account the electrostaticinteractions between the hydrophilic part of the lipidsdeposited on the air-water interface and the chargedmacromolecules at the subphase such as DNA10 andproteins.11-13 In this paper we study the interactions ofcholesterol and stearic acid with different chitosans at theair-water interface by analyzing the influence of theseinteractions on the isotherms produced by the interactionsof these lipids and the chitosans. To try to understand thechitosan-colesterol and chitosan-stearic acid interactions,we simulate the stomach conditions by using acidic chitosansolutions at a pH around 3. Langmuir-Blodgett films of themixtures were analyzed by atomic force microscopy (AFM),and a molecular simulation model was proposed aiming toglean understanding of the lipid-chitosan interactions. Theadsorption of cholesterol in powdered chitosans has also beenaddressed in this study by means of HPLC.

Experimental Methods

The cholesterol and stearic acid used were reagent gradeand obtained from Sigma (St. Louis, MO, U.S.A.); sodiumcyanoborohydride, chloroform, acetone, ethanol, dodecylaldehyde, and acetic acid were reagent grade and obtainedfrom Aldrich (Aldrich Chemical Co., Inc., Milwaukee, WI,U.S.A.); and methanol, hexane, and 2-propanol were HPLCgrade and obtained from Sigma also. Hydrochloric acid andsodium hydroxide were reagent grade and obtained fromMerck (Merck, Darmstadt, Germany). The water usedthroughout was of 18.3 MΩ‚cm and obtained from an EasyPure instrument from Barnstead. Chitosans of high (HMWchitosan) and medium molecular weights (MMW chitosan)were obtained from Aldrich (lot nos. 115080H0 and 05405CO,respectively, Aldrich Chemical Co., Inc., Milwaukee, WI,U.S.A.). The chitosan used to prepare the chloride salt wasobtained by thermoalkaline deacetylation of a chitin batchpreviously isolated from shrimp shell and head waste at apilot plant facility of Centro de Investigacio´n en Alimenta-cion y Desarrollo (C.I.A.D, Hermosillo, Sonora, Me´xico).

Chitosan chloride was obtained from a 1% (w/w) chitosansolution with 0.1 M HCl by precipitating the polymer withacetone. The solid was separated from the supernatant bycentrifugation and washed with different water/acetonemixtures and finally with pure acetone. The solid was driedfor 5 days at room temperature.

The hydrophobically modified chitosan (HYPM chitosan)was prepared by reductive amination of the MMW chitosanin homogeneous conditions usingn-dodecyl aldheyde.14 Twograms of chitosan were completely dissolved in 110 mL of0.2 M acetic acid and stirred. To this solution was added 75mL of ethanol to allow the aldehyde used for the alkylationto be solvated. The pH was adjusted to 5.1 to avoidprecipitation and a mixture of aldehyde-ethanol was addedto the appropriate ratio prior to an excess of sodium

cyanohydroborate (3 mol per chitosan monomole). Themixture was stirred for 24 h at room temperature. Thealkylated chitosan was precipitated with ethanol, and the pHwas maintained at 7 with a NaOH solution. The precipitatewas washed with several mixtures of water/ethanol, each timeincreasing the ethanol concentration from 70% (v/v) to 100%.

Molecular weights of the chitosan samples were obtainedby intrinsic viscosity measurements in 0.3 M acetic acid/0.2M sodium acetate at 25°C. The Mark-Houwink constantswere taken from the work of Rinaudo et al.15 The HMWchitosan molecular weight was 266 832, the MMW chitosanmolecular weight was 102 187, and the corresponding onefor chitosan chloride was 3500.

MMW chitosan, HMW chitosan, and HYPM chitosan usedas subphase solutions in the Langmuir balance experimentswere dissolved in 0.2 M HCl. Chitosan chloride solutionswere prepared in 18.3 MΩ‚cm water.

A Langmuir balance (Nima, Langmuir-Blodgett trough,model type 611, Coventry, U.K.), whose surface tensionprecision is 0.1 mN/m, was used to determine two-dimensional isotherms of cholesterol and stearic acid at theair-water interface. Different concentrations of chitosan andchitosan chloride were used as the subphase as mentionedabove, namely, 0.01, 0.02, and 0.03% (w/w). The pH of allthe chitosan solutions was adjusted to 3.1. Cholesterol andstearic acid solutions were prepared in chloroform (1 mg/mL) as solvent and used to be deposited on the water surfacewith a Hamilton microsyringe; 50µL of stearic acid and 30µL of cholesterol were deposited to build the correspondingisotherms. The temperature was kept at 20( 0.1 °C. Thecompression rate was taken at 50 cm2/min.

Calculations of the compressional modulus (Cs-1) were

performed with the OriginPro-7 (OriginLab. Co., Northamp-ton, MA, U.S.A.) software by interpolating theπ-A curvesby using 100 points obtained at equally spaced molecularareas along theπ-A isotherm.

AFM images of Langmuir-Blodgett samples depositedon mica were obtained in a JEOL instrument (scanning probemicroscope, JEOL, model JSPM 4210, Japan) in the non-contact mode, by using a NSC15 silicon cantilever from theMikroMasch Co. (MikroMasch, Portland, OR, U.S.A.).

The DA was determined by1H NMR, using a RMNinstrument (Avance 400 MHz, Bruker Co., Karlsrue, Ger-many). A total of 10 mg of the different chitosans used weredissolved in 0.5 mL of 2% (v/v) DCl/D2O. Samples werecompletely dissolved at 70°C. The DA was determinatedfrom the integral of theCH3 signal at 1.97 ppm comparedwith the integral of the H-I protons considered as an internalstandard.14 The DA obtained for the samples was 27% forthe HYPM chitosan, 16% for the HMW chitosan, 18% forthe MMW chitosan, and 22% for the chitosan chloride.

The interaction between chitosan and cholesterol or stearicacid was probed by an experiment by virtue of which 0.03%(w/w) solutions of different chitosans were prepared. A totalof 2 mL of each solution was put in a vial mixed with 200µL of 1 mg/mL cholesterol or stearic acid solution dissolvedin chloroform. The mixtures were vigorously agitated during30 min. After that, the suspensions are dried in a vacuumchamber. The dried complexes were mixed with KBr and

Chitosan-Cholesterol and -Stearic Acid Interaction Biomacromolecules, Vol. 6, No. 5, 2005 2417

measured in a Perkin-Elmer FT-IR system spectrum GX(Perkin-Elmer, Norwalk, CT, U.S.A.)

Molecular simulations were performed with the softwareGaussian 9816 in a Compaq Tru64 UNIX, V5.1 computer.The semiempirical PM3MM17 method was applied for theoptimization of the monomeric complexes, and after that,the UFF18 force field was used to optimize the polymericcomplexes of chitosan-cholesterol and chitosan-stearicacid.

Chitosan molecules were simulated in an acidic mediumby adding a proton to the-NH3

+ group. A cholesterol andor a stearic acid molecule was considered for each-NH3

+

group in the chitosan molecule.Adsorption studies were conducted at 25°C using 0.1 g

of each one of the four types of chitosan with two differentcholesterol concentrations (3 and 5 g/L) in 10 mL ofchloroform.

The extent of adsorption was calculated on the basis ofthe difference between the cholesterol concentrations in thesolution before and after adsorption. The cholesterol con-centration was determined by HPLC (Agilent HP model1100, U.S.A.) using a Zorbax Eclipse XDB-C18 reversephase column (Agilent Technologies, U.S.A.) at 35°C. Amobile phase consisting of solvents A (methanol) and B(hexane/2-propanol, 4:5, v/v) was used.

Results

Isotherms. In Figure 1 we show the isotherms ofcholesterol on a 0.2 M HCl water subphase and the isothermsof cholesterol with different chitosan solutions in thesubphase. The isotherms of cholesterol are very similar tothe ones found by other authors.19-22 Cholesterol gives atypical condensed monolayer with an extrapolated area (Aex)of 39.6 Å2/molecule (estimated by extrapolation of the high-pressure linear part of theΠ-A curve to zero surfacepressure) and an area of the “onset of condensation” (A0)(the area per molecule at a surface pressure of 1.5 mN/m)20

of 44.0 Å2. In Figure 1 we show the shift of the isothermsproduced by the different chitosans at the same concentration(0.01%, w/w) in the subphase and the cholesterol at the air-

water interface. There is noticed a great variation inAex foreach chitosan due to differences in the interactions withcholesterol at the air-water interface. For example, weobserve anA0 ) 64.9 Å2 for the HYPM chitosan indicatingan average difference of 64.9- 44 Å2 ) 20.9 Å2 of emptyarea/molecule for the HYPM chitosan compared with thecholesterol alone when the pressure starts rising (1.5 mN/m). When the pressure is increased at the condensed phase,the difference inAex reaches 48.0- 39.6 ) 8.4 Å2. Thismeans that now this phase is less condensed than that ofcholesterol alone and also that the magnitude of this emptyarea is depending on the type of cholesterol-chitosaninteraction. As can be seen in Figure 1, theA0 and theAex

are lower for the chitosan chloride compared with the otherchitosans analyzed. This could be diagnostic that the interac-tion of cholesterol-chitosan chloride is weak compared withthe other macromolecules and that the low molecular sizeof chitosan chloride is affecting the compactness of themonolayer.

In Figure 2 is shown the effect of the concentration ofMMW chitosan in the subphase on the isotherm of choles-terol. Inspection of the figure shows that as the concentrationof chitosan increases the values ofAex andA0 also increases,thus, effectively indicating that when more chitosan mol-ecules are added to the subphase, the cholesterol moleculesjoined to more macromolecules or more sites in one chitosanmolecule become more separated. This separation amongcholesterol molecules persists even when pressure is in-creased and the molecular area is reduced, indicating thatthe molecular bonds are not lost at higher pressure. The slopeof the curves is reduced as the chitosan concentrationincreases, indicating a decrease of the compressional modulus(Cs

-1) as can be seen in Table 1. In Table 1 are summarizedtheAex, A0, andCs

-1 values for the isotherms of cholesterolwith the different chitosans in the subphase. We observe thatfor chitosan chloride experiments, only a small variation inAex andA0 for all concentrations is noticed and the isothermsremain almost joined and separated only a little from theone of cholesterol alone (results not shown). HYPM chitosanshowed the maximum shift in the isotherms in the condensedphase, and HMW chitosan showed the maximum shift in

Figure 1. Isotherms of cholesterol and cholesterol-chitosan on theair/water interface. The different chitosans were 0.01% (w/w) dis-solved in a 0.2 M HCl water solution.

Figure 2. Isotherms of cholesterol and cholesterol-MMW chitosanon the air/water interface. Three different MMW chitosan concentra-tions were dissolved in a 0.2 M HCl water solution.

2418 Biomacromolecules, Vol. 6, No. 5, 2005 Parra-Barraza et al.

the “onset area” as observed from the values ofAex andA0,respectively. At higher pressure, this could be understoodas a contribution of the hydrophobic groups linked to the

chitosan molecules that compete with cholesterol at the air-water interface. At lower pressure, the effect of the stericimpediment exerted by larger chitosan molecules is moreimportant than the hydrophobicity of the HYPM chitosan.

The behavior ofCs-1 showed interesting differences with

the type of chitosan and the concentration. The maximumvalue ofCs

-1 was reached at the lower concentration usedfor all chitosans and from all materials, and the MMWchitosan showed the maximum values ofCs

-1. The resultsindicate that the rigidity of the cholesterol monolayer is beingaffected by the chitosan interactions, and when the concen-tration is increased, the monolayer becomes more flexible.Another interesting parameter found for the behavior ofCs

-1

was the area corresponding at the maximum of the compres-sional modulus (AM). As shown in Table 1, this area waslower than the correspondingA0 for each isotherm and itwas very near the collapse point. Very little difference was

Table 1. Extrapolated Area (Aex), Area of the “Onset ofCondensation” (A0), and Maximum Compressional Modulus (Cs

-1)of the π-A Isotherms at the Molecular Area AM of Cholesterol andCholesterol-Chitosan Isotherms at the Air-Water Interface forDifferent Chitosan Concentrations

complexcholesterol-

chitosan(%, w/w)

A0

(Å2)Aex

(Å2)Cs

-1

(mN/m)AM

(Å2)

cholesterol 0 44.0 39.6 417 41.6HYPM chitosan 0.01 64.9 48.0 272 46.1

0.02 69.2 55.8 207 51.00.03 76.6 61.5 191 52.8

HMW chitosan 0.01 58.3 55.5 233 53.10.02 60.6 56.9 187 52.70.03 71.5 68.9 160 53.5

MMW chitosan 0.01 54.0 51.4 282 49.10.02 59.8 56.7 202 50.70.03 71.2 67.2 133 60.1

chitosan chloride 0.01 49.5 47.5 255 43.70.02 51.5 49.5 229 43.00.03 51.5 49.8 254 43.4

Figure 3. Isotherms of stearic acid and stearic acid-chitosans onthe air/water interface. The different chitosans were 0.01% (w/w) anddissolved in a 0.2 M HCl water solution.

Figure 4. Isotherms of stearic acid and stearic acid-MMW chitosanon the air/water interface. Three different MMW chitosan concentra-tions were dissolved in a 0.2 M HCl water solution.

Table 2. Extrapolated Area (Aex), Area of the “Onset ofCondensation” (A0), and Maximum Compressional Modulus (Cs

-1)of the π-A Isotherms at the Molecular Area AM of Stearic Acidand Stearic Acid-Chitosan Isotherms at the Air-Water Interfacefor Different Chitosan Concentrations

complexstearic acid-

chitosan(%, w/w)

A0

(Å2)Aex

(Å2)Cs

-1

(mN/m)AM

(Å2)

stearic acid 0 28.9 22.5 407 22.8HYPM chitosan 0.01 35 26.1 207 23.0

0.02 44.2 33.7 150 27.90.03 51.2 39 103 31.9

HMW chitosan 0.01 34.7 31.8 181 26.20.02 39.2 35.8 143 28.50.03 41.6 35.8 138 30.1

MMW chitosan 0.01 41.1 29.1 176 29.40.02 41.6 31.1 145 29.90.03 42.8 34.6 144 28.9

chitosan chloride 0.01 31.4 23.7 247 23.40.02 31.4 26.3 302 25.20.03 33.6 27.9 198 25.4

Table 3. Maximum Peak Heights of the Surfaces Observed fromthe Height Histograms and rms Roughness Measurements forStearic Acid, Cholesterol, Different Chitosans, and Their MixturesObtained on Mica

samplechitosan(%, w/w)

maximumpeak (nm)

roughness(nm)

cholesterol 0 1.13, 1.45 0.4stearic acid 6.0, 21.3 9.6HYPM chitosan 0.01 3.5, 18.7 4.65

0.03 34, 42 5.67HYPM chitosan-cholesterol 0.01 44.5, 197 66

11.9, 37.5 9.31HYPM chitosan-stearic acid 0.03 2.3, 28.0 8.71

4.3 2.74

HMW chitosan 0.01 2.17, 9.93 4.00.03 0.8, 5.7 2.87

HMW chitosan-cholesterol 0.01 2.01, 10.0 3.14.13, 20.0 5.5

HMW chitosan-stearic acid 0.03 10.39 1.832.2, 16.7 7.2

MMW chitosan 0.01 1.24, 5.6 1.50.03 31, 45.6 12.3

MMW chitosan-cholesterol 0.01 1.56, 4 1.351.6, 2.0 1.41

MMW chitosan-stearic acid 0.03 1.6, 10.8 7.72.2, 6.6 6.1

Chitosan-Cholesterol and -Stearic Acid Interaction Biomacromolecules, Vol. 6, No. 5, 2005 2419

found for theAM values of chitosan chloride with respect tothe one of pure cholesterol. Our value ofCs

-1 for purecholesterol resulted higher than the one reported by Go´mez-Serranillos et al.21 The maximum value they found wasCs

-1

) 289 mN/m at 10 mN/m. However, they did not mentionthe numerical method used in the calculation. It seems thatthe Cs

-1 values obtained by different researchers differdepending on the method used. Li et al.21 have pointed outthe importance of the method used to compute compressionalmodulus values of monolayers.

Our results of the interaction of stearic acid and thedifferent chitosans at the lower concentration (0.01%, w/w)analyzed are shown in Figure 3. The monolayer of stearicacid exhibits the typical isotherm with a change of slope atπ ) 24 mN/m at the transition from the liquid condensed tothe solid state, similar to the value obtained by Seoane etal.19 The extrapolated area of stearic acidAex yields 22.5Å2, similar to the value found by them too. However, thecollapse pressure obtained by us was lower than the oneobtained by these authors, probably due to differences of

the compression rate. The collapse pressure is obtained whenthe isotherm exhibits a sharp peak after the solid phaseappears, followed by a drop of surface tension,19 and hasreceived some attention due to the importance to understandthe aggregation phenomenon when the monolayer is com-pressed until it starts to destroy the two-dimensional struc-ture.23

In Figure 3 we show the shift of the isotherms producedby the different chitosans at the concentration 0.01% (w/w)in the subphase and the stearic acid at the air-water inter-face. Notice that a lower variation inAex was observed amongthe different chitosans as compared to the shift producedwith cholesterol with the same chitosans, presumably dueto the structural differences between cholesterol and stearicacid interacting with chitosans. We observe anA0 ) 35 Å2

for the HYPM chitosan indicating a difference of 35-28.9 Å2 ) 6.1 Å2, comparing with stearic acid alone. Whenthe pressure is increased at the condensed phase, thedifference in Aex reaches 26.1- 22.5 ) 3.6 Å2. Thesedifferences are smaller compared with the ones obtained with

Figure 5. AFM images of mixtures of cholesterol and MMW chitosans for two different chitosan concentrations. (a) MMW chitosan directlyobtained with the dipper from the aqueous solution, (b) cholesterol, (c) cholesterol-0.03% (w/w) MMW chitosan, and (d) cholesterol-0.01%(w/w)MMW chitosan.

2420 Biomacromolecules, Vol. 6, No. 5, 2005 Parra-Barraza et al.

the same chitosan and cholesterol, indicating that the averagedistance among cholesterol molecules is larger than the oneobserved with stearic acid with the same chitosan in thesubphase. As can be seen in Figure 3, theA0 and theAex arelower for the chitosan chloride compared with the otherchitosans analyzed. This could mean, similarly as in the caseof cholesterol, that the interaction of stearic acid-chitosanchloride is much weaker when compared with the othermacromolecules.

In Figure 4 we show the effect of the concentration ofMMW chitosan in the subphase on the isotherm of stearicacid. A slight increasing effect of the concentration ofchitosan on theAex and A0 is observed, thus, effectivelyindicating that the more chitosan molecules are added to thesubphase, the more separated remain the stearic acidmolecules joined to more macromolecules or more sites perchitosan molecule. However, the effect is less significantcompared with the one shown with the same chitosan and

cholesterol. In this case we observe that the collapse in theisotherms starts at lower pressure and the solid phase is veryshort.

The Aex, A0, andCs-1 values for the isotherms of stearic

acid with the different chitosans in the subphase are presentedin Table 2. We observe again that chitosan chloridedemonstrates a small shift ofAex andA0 and the isothermsremain separated only a little from the one of stearic acidalone (results not shown). HYPM chitosan showed themaximum shift in the isotherms in the expanded phase asobserved from the values ofA0, hence, showing its highaffinity with stearic acid.

The behavior ofCs-1 of the monolayers with chitosans

and stearic acid showed behavior similar to that observedwith cholesterol. The maximum value ofCs

-1 was reachedat the lower concentration used for all chitosans, and ingeneral the attained values ofCs

-1 were lower whencompared to the ones obtained for the same chitosan and

Figure 6. AFM images of two different chitosan-cholesterol mixtures obtained from the Langmuir balance. (a) 0.03% (w/w) HYPM chitosan-cholesterol, (b) height histogram of HYPM chitosan-cholesterol, (c) 0.03% (w/w) HMW chitosan-cholesterol, and (d) height histogram of theHMW-chitosan mixture.

Chitosan-Cholesterol and -Stearic Acid Interaction Biomacromolecules, Vol. 6, No. 5, 2005 2421

cholesterol shown in Table 1. The behavior ofAM for thestearic acid-chitosans was similar to the one shown forcholesterol-chitosans. The results indicate that stearic acid-chitosan monolayers show more flexibility than the corre-sponding ones with cholesterol.

IR Analysis. Analysis of the chitosan-stearic acid inter-action was performed by IR spectroscopy. Two characteristicbands centered at 1639 and 1516 cm-1 were clearly presentcorresponding to vibrations of the NH3 group in chitosan. Aband located at 1095 cm-1 corresponding to the pyranosering was also observed. Bands characteristic of stearic acidwere observed at 1707 cm-1 assigned to vibrations of theCOOH group. Another band was observed at 1618 cm-1

corresponding to the group COO-. These results are inagreement with the ones found by Demarger et al.24 Samplesof cholesterol-chitosan did not show evidence of changesin the chitosan spectra.

AFM Measurements. Langmuir-Blodgett films on freshlycleaved mica were obtained at 15 mN/m from the Langmuir

balance and observed by AFM. Different films of chitosans,cholesterol, stearic acid, and their complexes formed at theair-water interface were obtained when different chitosansolutions were used as the subphase.

In Figure 5 are shown the images of MMW chitosan,cholesterol, and the complexes off cholesterol-MMW chi-tosan for two different chitosan concentrations. In Figure 5a,a MMW chitosan film is included; inspection of the AFMmicrograph reveals the presence of large aggregates (30.1nm average height) with a root-mean-square (rms) roughnessof 12.3 nm. The cholesterol monolayer observed in Figure5b was similar to the one reported by other authors. A rmsroughness of 0.4 nm and an average height of 1.56 nm weremeasured, which is in good agreement with the valuereported by Kim et al.11 (1.6 nm). The film obtained withcholesterol at the air-water interface and MMW chitosan(0.03%, w/w) is displayed in Figure 5c. In this case, a morehomogeneous surface with a rms roughness of 7.7 and 4.95nm of average height is present, but still we notice the

Figure 7. AFM images of mixtures of stearic acid-MMW chitosan for two different chitosan concentrations. (a) Stearic acid, (b) 0.03% (w/w)MMW chitosan-stearic acid, (c) 0.01% (w/w) MMW chitosan-stearic acid, and (d) height histogram of 0.01% MMW chitosan-stearic acid.

2422 Biomacromolecules, Vol. 6, No. 5, 2005 Parra-Barraza et al.

presence of some domains of 1.6 nm height correspondingto the average height of pure cholesterol. Figure 5d showsthe AFM image of the same complex at a lower chitosanconcentration (0.01%). The measured roughness was lower(1.35 nm), and the average height decreased to 4 nm,showing domains of 1.6 nm height corresponding to cho-lesterol. These results indicate that the mixture of cholesteroland chitosan on the mica surface is more homogeneous thanthe chitosan alone due to the chitosan-cholesterol interac-tion. The excess of chitosan produces a more roughenedsurface intercalated with regions of pure cholesterol. Thehigher peaks of the height histograms and the rms roughnessare shown in Table 3 for all samples and chitosan concentra-tions analyzed.

The effect of the different sizes of chitosan molecules andthe addition of a short hydrophobic chain on the chitosanmolecule are shown in Figure 6. The film obtained with themixture HYPM chitosan-cholesterol showed a wide rangeof heights, with two peaks of 2.3 and 28 nm and a rmsroughness of 8.71 nm. As observed in Figure 6a, the image

shows small aggregates corresponding to HYPM chitosanand the smallest ones to cholesterol. The correspondingheight histogram is shown in Figure 6b. The films of HMWchitosan-cholesterol are shown in Figure 6c, and thecorresponding height histogram is in Figure 6d. As observed,the surface shows a more homogeneous surface with arms roughness of 1.83 nm. The average heights of bothHMW and MMW chitosan-cholesterol surfaces are similar(around 10 nm), and no evidence of isolated domains ofcholesterol were found, showing a more homogeneousmixture compared to the one of the HYPM chitosan-cholesterol mixture.

The films obtained with stearic acid and different chitosansare shown in Figure 7. This figure corresponds to the stearicacid film, showing a rms roughness of 9.6 nm probably dueto crystal formation. The height distribution showed twopeaks at 2 and 21.9 nm. The first value corresponds to thesize of one monolayer of stearic acid on the mica, and theother corresponds to multilayers as previously observed atthe collapse pressure by Valdez-Covarrubias et al.23

Figure 8. AFM images of two different chitosans mixed with stearic acid obtained from the Langmuir balance. (a) 0.03% (w/w) HYPM chitosan-stearic acid, (b) height histogram of HYPM chitosan-stearic acid, (c) 0.03% (w/w) HMW chitosan-stearic acid, and (d) height histogram of theHMW-chitosan mixture.

Chitosan-Cholesterol and -Stearic Acid Interaction Biomacromolecules, Vol. 6, No. 5, 2005 2423

In the film of MMW chitosan-stearic acid shown inFigure 7b, smaller aggregates are observed, compared withthe ones found in MMW chitosan alone (Figure 5a). Some

domains of 2.3 nm height around the chitosan aggregatesare also evident due to the stearic acid monolayer. Themeasured roughness was 6.15 nm, and the peak height was6.6 nm corresponding to the chitosan aggregates. Observethat the surface, similarly to the case with cholesterol, is morehomogeneous than the one with chitosan alone. When thechitosan concentration was lowered, we measured a loweraverage height of 3.8 nm (Figure 7d) and a lower rmsroughness of 1.4 nm. Again, as can be noticed in Figure 7c,zones of pure stearic acid of average height around 2.3 nmare observed. If we compare these images with the corre-sponding ones by using cholesterol (Figure 5), we noticethat a greater proportion of isolated domains of stearic acidand MMH chitosan is present.

The films of different chitosans and stearic acid are shownin Figure 8. As observed in Figure 8a, the surface of themixture HYPM chitosan-stearic acid seems to be more

Figure 9. Molecular simulations in the bulk of the complexes of chitosan-cholesterol and chitosan-stearic acid obtained with the Gaussian 98software and using four monomers of chitosan: (a) chitosan-cholesterol and (b) chitosan-stearic acid.

Table 4. Adsorbed Cholesterol on Different Chitosans by HPLCa

sample

cholesterolinitial

concentration(g/L)

adsorbedcholesterol/chitosan

(g/g)% adsorbedcholesterol

MMW chitosan 3 0.098 33HMW chitosan 3 0.085 28chitosan chloride 3 0.039 13HYPM chitosan 3 0 0MMW chitosan 5 0.127 42HMW chitosan 5 0.115 38chitosan chloride 5 0.023 8HYPM chitosan 5 0.009 3

a Two initial cholesterol-in-chloroform concentrations were used, anda 10 mL solution was mixed with each chitosan sample (0.1 g).

2424 Biomacromolecules, Vol. 6, No. 5, 2005 Parra-Barraza et al.

homogeneous, as compared to the one obtained in the sameconditions with the same chitosan and cholesterol (Figure6a) with an average height of 4.3 nm (Figure 8b) and a rmsroughness of 2.7 nm. On the contrary, for the case of HMWchitosan with stearic acid (Figure 8c,d), the images show amore heterogeneous surface than the corresponding oneobtained with cholesterol, with a roughness of 7.9 nm, similarto the one obtained with MMW chitosan-stearic acid. Theheight distribution shown in Figure 8d, for the film of HMWchitosan-stearic acid, presents two peaks of 2.19 and 16nm corresponding to the stearic acid monolayer and thechitosan aggregates, respectively. The effect of the molecularweight of chitosan can clearly be appreciated when compar-ing Figure 8c with Figure 7b, where the MMW chitosan-stearic acid film showed a height distribution with two peaksof 2.2 and 6.6 nm due to a pure stearic acid monolayer andsmaller MMW chitosan aggregates than those of HMWchitosan.

Molecular Simulations. The chitosan molecule was builtconsidering a DA of approximately 20% and without takinginto account the interface. First, the chitosan monomer wasoptimized with four units ofD-glucosamine and one unit ofN-acetyl-D-glucosamine using the PM3MM17 method tosearch the available electrostatic bonds between chitosan andcholesterol and between chitosan and stearic acid. Once themonomeric complex was optimized, we tested the interactionof four monomeric units of chitosan with cholesterol andstearic acid.

In Figure 9a we show an image of the chitosan-cholesterol complex. This structure is similar to the oneobtained for chitosan by Kim et al.,25 using similar methods.The structure found for the chitosan was similar to thechitosan without the hydrophobic group, and it seemed thatcholesterol did not cause great perturbation in the shape ofthe chitosan molecule. The interaction between cholesteroland chitosan is achieved between the OH group of cholesteroland several charged sites of the chitosan molecule (-NH3

+).Measuring the separation between two union sites, wherecholesterol can interact with the chitosan molecule, weobtained a value of∼9 Å. This value is in excellentagreement with data from Table 1, where we obtained theaverage distance between cholesterol molecules at theextrapolated area of around 8 Å and a maximum value of9.3 Å. This could give us an idea that the nearest cholesterolmolecule from one reaction site of a chitosan moleculeprobably belongs to the same chitosan chain.

In Figure 9b we observe the result of the interactionbetween chitosan and stearic acid. The chitosan moleculewas simulated in acidic conditions, and the amine groupsalong the whole chitosan chain were protonated; conse-quently, the electrostatic interactions were formed with the-NH3

+ of chitosan and the COO- group of the stearic acid.Similar results were found recently by Thongngam andMcClements26 investigating the interaction between chitosanand an anionic surfactant (sodium dodecyl sulfate). Theydemonstrated that the interaction is electrostatic and isstronger at a pH around 3.

The measured distance between a reactive site and the nextone of a stearic acid molecule in the chitosan was around

9.4 Å (Figure 9b). In Table 2 from the extrapolated area ofdifferent chitosan-stearic acid complexes, we obtained aminimum average distance between stearic acid moleculesaround 6 Å and a maximum value of 7 Å. This could meanthat the nearest stearic acid molecule to the one chosenprobably is bounded to a different chitosan molecule due tothe excess of pressure in the stearic acid monolayer.

HPLC Measurements.The results of cholesterol adsorp-tion on powdered chitosans measured by HPLC are shownin Table 4. The values of the adsorbed cholesterol were ofthe same order but lower compared to the ones reported byChiu et al.27 using cyclodextrin bounded to the chitosanchain. This is consistent with the proposal that the nature ofthe molecular group attached to the chitosan chain is veryimportant to enhance the cholesterol affinity of chitosans.Interesting is the result for HYPM chitosan, which did notshow evidence of significant cholesterol adsorption. Thisbehavior could be explained as follows: in the HPLCexperiments, the interaction between cholesterol and theHYPM chitosan is performed between a solid surface and asolution of cholesterol. We assume that the hydrophobicgroups of this chitosan remain at the surface, and this couldpreclude the interaction of cholesterol with-NH3

+ groups,producing a very low attraction due to a steric hindrancemechanism. On the other hand, the shift of the isotherms ofcholesterol with HYPM chitosan as the subphase (Figure 1)was enhanced by the presence of the alkyl groups of themodified chitosan at the air-water interface presumably asa consequence of a strong cholesterol-HYPM chitosaninteraction.

Conclusions

In this work we have investigated the interaction ofcholesterol and stearic acid with different chitosans at theair-water interface. Our results showed great modificationsof the isotherms and good stability of the monolayers ofcholesterol and stearic acid interacting with chitosans atdifferent concentrations in the subphase. By measuring theextrapolated area and the area of the “onset condensation”of the different samples, we conclude that cholesterol andstearic acid molecules remain attached at specific sites ofchitosan molecules during the compression cycle of theisotherm, producing highly spaced cholesterol and stearicacid molecules in the interface. The film rigidity of themonolayers, for both cholesterol and stearic acid, wasstrongly affected with the interaction of chitosans producingmore flexible films. The AFM measurements of the Lang-muir-Blodgett samples of the mixtures of cholesterol-chitosan and stearic acid-chitosan indicated that cholesterolhad better affinity with both HMW and MMW chitosans butnot with HYPM chitosan, a result that could be correlatedwith the negligible adsorption of cholesterol in powderedHYPM chitosan. On the contrary, stearic acid, according tothe AFM measurements, showed more affinity with HYPMchitosan compared with the separated domains observed forHMW and MMW chitosans. Finally, the simulations of thecomplexes of chitosan-cholestrol and chitosan-stearic acidin bulk and without taking account other conditions sug-

Chitosan-Cholesterol and -Stearic Acid Interaction Biomacromolecules, Vol. 6, No. 5, 2005 2425

gested, according to the results of the isotherms, that thenearest cholesterol molecule to one attached to a chitosanmolecule at the air-water interface probably belongs to thesame chitosan molecule.

Acknowledgment. We appreciate the financial supportof C.O.N.A.C. y T., Mexico, under Grant ER074 “MaterialesBiomoleculares”.

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