Structure/Function Relationships of Several Biopolymers...

Structure/Function Relationships of Several Biopolymers asRelated to Invertase Stability in Dehydrated Systems

Patricio R. Santagapita,† Leissy Gómez Brizuela,‡ M. Florencia Mazzobre,†

Héctor L. Ramirez,‡ Horacio R. Corti,§,4 Reynaldo Villalonga Santana,‡ andM. Pilar Buera*,†

Departamentos de Industrias y de Química Orgánica, Facultad de Ciencias Exactas y Naturales,Universidad de Buenos Aires, Ciudad Universitaria 1428, Buenos Aires, Argentina, Centro de Estudios deTecnología Enzimática, Facultad de Agronomía, Universidad de Matanzas “Camilo Cienfuegos”, Autopista

a Varadero km 3½, 44740, Matanzas, Cuba, Comisión Nacional de Energía Atómica, Centro AtómicoConstituyentes, Av. Constituyentes, San Martín, Buenos Aires, Argentina, and Departamento de QuímicaInorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos

Aires, Ciudad Universitaria 1428, Buenos Aires, Argentina

Received November 2, 2007; Revised Manuscript Received December 3, 2007

Structure/function relationships of different biopolymers (alginate, dextran, or �-cyclodextrin) were analyzed assingle excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-driedformulations and compared to trehalose. Particularly, a novel synthesized polymer �-cyclodextrin-branched alginate(�-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer betterprotection to invertase compared to trehalose. �-CD-A (with or without trehalose), �-cyclodextrin (�-CD), ordextran with trehalose were the best protective agents during thermal treatment, while �-CD and alginate showeda negative effect on invertase activity preservation. The �-CD linked alginate combined the physical stabilityprovided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. �-CD-Awas effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combinationof two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins,modifying enzyme stability patterns.

1. Introduction

The use of enzymes at the industrial level is often limited bythe low resistance of these proteins to technological conditions,especially at high temperatures. Important efforts based on site-direct mutagenesis,1 immobilization on solid supports,2 use ofwater-soluble additives,3 and chemical,4–7 enzymatic,8 andchemoenzymatic9 modifications of the protein surface have beenreported to improve enzyme thermostability. From the industrialpoint of view, the use of hydrosoluble additives appears as oneof the most promising approaches, considering the ease and lowcost of this procedure.10

Alginates and dextrans are employed as ingredients, excipi-ents, or protein stabilizers in food and pharmaceutical industriesor for biotechnological purposes (immobilization media forenzyme or bacteria/preparation of hydrogels).11–15 Alginate (A)is a binary copolymer of (1f4) linked �-D-mannuronic and itsC5 epimer (R-L-guluronic acid) residues, negatively charged atpH values higher than 3.65.16 Dextran (D) is a D-glucose linearpolymer composed of 95% R(1f6) linkage, is mostly unchargedin a wide pH range, and behaves as a very flexible and extended

polymer. Although they are widely used, it has been demon-strated that they are not as good as trehalose (T) to protectenzymes after freeze-drying or thermal treatment.17,18 It is well-known that the water replacement mechanism is one of the keysof the success of trehalose as a cryo- and dehydroprotectant oflabile systems.19 However, undesired trehalose crystallizationprocesses have become a main issue in pharmaceutical and foodingredients industries because crystallization of the solid phasesignicantly affects the mechanisms by which amorphous sugarsmanifest protective effects on biomolecules and, consequently,the shelf life of a product.20–23 Several authors have demon-strated that the presence of a polymer delays sugar crystallizationprocesses.20,21,24–27

Another approach for increasing protein stability in dehy-drated formulations is the use of cyclodextrins. �-Cyclodextrin(�-CD) is a cyclic nonreducing oligosaccharide composed ofseven glucopyranose units bonded together via R(1–4) glycosidelinkages, with a hydrophobic central cavity and a hydrophilicouter surface.28 CDs are capable of including a variety ofhydrophobic guest compounds such as aromatic amino acidslocated at the proteins surface.4,5,8 Thermal stability of enzymescould thus be improved by chemical conjugation of the enzymewith several CD derivatives that induced multipoint supramo-lecular advantageous interactions at the surface of the enzyme.4,5,8

On the other side, covalently modified biopolymers (polysucroseor dextran) with cyclodextrins were used as excipients forimproving enzyme protection in very diluted aqueous media.10,29

Also, in diluted systems, a modification of alginate with R-cyclodextrin generated through the addition of the CD moieties

* Author to whom correspondence should be addressed. E-mail: [email protected]. Telephone/Fax: +(54) 114576-3366.

† Departamentos de Industrias y de Química Orgánica, Facultad deCiencias Exactas y Naturales, Universidad de Buenos Aires. Telephone/Fax: +(54) 114576-3366.

‡ Centro de Estudios de Tecnología Enzimática, Facultad de Agronomía,Universidad de Matanzas “Camilo Cienfuegos”. Telephone: 5345 262251.Fax: 5345 253101.

§ Comisión Nacional de Energía Atómica, Centro Atómico Constituyentes.4 Departamento de Química Inorgánica, Analítica y Química Física,

Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires.

Biomacromolecules 2008, 9, 741–747 741

10.1021/bm7012108 CCC: $40.75 2008 American Chemical SocietyPublished on Web 01/12/2008

in the alginate secondary hydroxyl groups was tested in regardof its host–guest complex ability.30

In the present work, structure/function relationship of differentbiopolymers were related to their efficiency as enzyme stabiliz-ers in freeze-dried formulations and compared to trehalose invery low water content environments. Biopolymers such asalginate and dextran, and particularly a synthesized �-CD-branched alginate (�-CD-A), modified through the carboxylicalginate groups, were employed. �-CD-A was designed tocombine the physical stability provided by alginate with theprotective effect on hydrophobic regions of the enzyme providedby cyclodextrin.

2. Materials and Methods

2.1. Synthesis of �-Cyclodextrin-Ethylenediamine-AlginatePolymer. �-CD was purchased from Amaizo (Hammond, IN). Mono-6-(ethylenediamine)-6-deoxy-�-CD was previously obtained by treatingmono-6-O-tosyl-�-CD31 with ethylenediamine in aqueous solution.32

The product was purified by cation exchange chromatography on CM-Sephadex C-25 (NH4

+ form) and characterized by conventional NMRtechniques. To introduce the �-CD derivative into the polysaccharidechains, 400 mg of �-CD derivative and 200 mg of sodium alginate(sodium alginate was obtained from Laminaria hyperborean, BDH,Poole, UK; molecular weight ) 1.97 × 105,33 mannuronate/guluronateratio ) 0.6)34 were dissolved in 10 mL of distilled water and then 120mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide were added. Thesolution was stirred for 16 h at 25 °C, dialyzed against distilled H2O,frozen at -26 °C for 24 h, and finally freeze-dried in a Heto HoltenA/S cooling trap model CT 110 freeze-dryer (Heto LaboratoryEquipment, Denmark) operating at a condenser plate temperature of-111 °C and a chamber pressure of 4 × 10-4 mbar. The secondarydrying was performed at 25 °C. The final product, �-CD-ethylenedi-amine-alginate (�-CD-A), was characterized by 1H NMR (500 MHzequipment (Bruker Optics, Rheinstetten, Germany) in solution of 20mg/cm3 in D2O at 25 °C) and GPC (Shimadzu L6A with columnsAsahipak GS series GS 220, 320, and 520, using a refraction indexdetector RID-6A, flux of 1.0 mL/min, 200 µL as a injected volume,buffer pH 7, ambient temperature, with a C-R4 Chromatopac GPCprogram as integrator). The calibration curve for gel permeationchromatography (GPC) was performed using the weight-averagemolecular weight (Mw) vs eluted volume, using patterns of pullulan inthe range of 5800-1 600 000 and sucrose.

2.2. Preparation of the Samples and Thermal Treatment. Solidsystems consisted of freeze-dried solutions containing 10% (w/v) ofR-R-trehalose dihydrate (T), (Hayashibara Co, Ltd., Shimoishii, Okaya-ma, Japan/Cargill Inc., Minneapolis, MN), the polymer (dextran 70 000,sodium alginate, �-CD-A, and �-CD) or the blend trehalose-polymer(1:1); in all systems, the enzyme invertase (�-fructofuranosidase, EC3.2.1.26) of Saccharomyces cereVisiae (1840 U/mg, Fluka, Buchs,Switzerland) was added with a dilution factor of 1/100 of a solution6.36 mg/cm3. One unit of activity was defined as the amount of enzymerequired to hydrolyze 1.0 µmol of sucrose per minute at pH 4.6 at 37°C. Invertase concentration was estimated spectrophotometrically byabsorbance at 280 nm, considering the relationship 1 mg/cm3 ) 2.25(absorbance unit).35

Aliquots of 0.075 cm3 of each model were placed in Eppendorf tubes,frozen by immersion in liquid nitrogen immediately before freeze-dryingin a Heto Holten A/S freeze-dryer (Heto Laboratory Equipment). Afterfreeze-drying, the samples were transferred to vacuum desiccators andexposed to relative vapor pressure (RVP) of 22% (saturated solutionof CH3COOK) and 43% (saturated solution of K2CO3) at 25 ( 1 °Cfor one week.36 After rehumidification, the samples were hermeticallysealed and stored at 87 ( 3 °C in a forced air convection oven. At asuitable interval, two samples were removed from the oven and theremaining activity of the enzyme was determined as describe below;the average value was reported.

2.3. Invertase Activity. The samples were resuspended in 0.75 cm3

of 50 mM sodium acetate buffer pH 4.6 and were kept at 5 °C untilcomplete dissolution was achieved. The enzymatic activity of invertasewas determined by adding 0.40 cm3 of sucrose solution (200 mM insodium acetate buffer 50 mM, pH 4.6) to 0.10 cm3 of the dissolutionsample. After 10 min at 37 °C, the reaction was stopped and thereducing sugars were determined spectrophotometrically at 546 nmusing 3,5-dinitrosalicylic acid method.37 For each system, the amountof hydrolyzed sucrose after a given time of thermal treatment (S) wasrelated to the amount of sucrose hydrolyzed before the thermal treatment(S0) and the remaining activity (RA) was expressed in percentage as:RA ) 100(S/S0). Duplicate measurements were performed for eachanalysis, and the confidence interval, calculated by measuring foursamples of the same run, was 7%.

2.4. Thermal Transitions. Glass-transition temperature (Tg) andendothermal peaks areas (related to the amount of crystalline trehalosedihydrate present in the samples) were determined by dynamicdifferential scanning calorimetry (DSC) by means of a Mettler Toledo822 equipment (Mettler Toledo AG, Switzerland) and STARe thermalanalysis system version 3.1 software (Mettler Toledo AG). For eachsystem, the endothermal baseline shift represents the glass transitionand the endothermal peak correspond to the melting of crystallinetrehalose dihydrate. Glass transitions were recorded as the onsettemperatures of the discontinuities in the curves of heat flow versustemperature. The instrument was calibrated using standard compounds(indium and zinc) of defined melting point and heat of melting. Allmeasurements were made in duplicate with 5–10 mg sample mass, usinghermetically sealed aluminum pans of 0.040 cm3 inner volume (Mettler),heated from -100 to 120 °C at 10 °C/min; an empty pan was used asa reference. The confidence interval estimated for temperature valueswas 1 °C.

In trehalose-containing systems, the degree of trehalose crystallization(�) was calculated from the ratio of the area of the endothermic meltingpeak in the sample thermogram (∆Hm) and the melting enthalpy ofpure trehalose dihydrate (∆HmT, 139 J/g) measured by dynamic DSCin the same conditions:

� ) ∆Hm ⁄ ∆HmT (1)

The confidence interval estimated for enthalpy values was 10 mJ.2.5. Atomic Force Microscopy (AFM). AFM images were taken

on a Multimode AFM with a Nanoscope IIIa controller (DigitalInstruments, Veeco, Santa Barbara, CA), using an AS-130 scanner (“J”,lateral range 125 µm × 125 µm, vertical range 5 µm) in contact mode.The cantilever was made out of SiN with a spring constant of about0.06 N/m and a nominal tip apex radius of 20 nm (DNP, fromNanoprobes, Veeco). Height and deflection data were collectedsimultaneously. Both images were collected with a force of ap-proximately 1 nN, and the scan rate was of 3.05 Hz (tip velocity 6.10µm/s); the size of the images was 1 µm × 1 µm, and the resolutionwas 512 × 512 pixels. All of the images are presented without anyfiltering or modification. For sample preparation, films were obtainedby a dip-coating procedure over thin glasses at a coating rate of 2mm/s.

2.6. Determination of Water Content. The total water content ofthe samples was determined gravimetrically by difference in weightbefore and after drying in a vacuum oven for 48 h at 96 ( 2 °C. Thesedrying conditions were selected in previous studies,38,39 and they wereadequate to determine water content in the studied systems with aconfidence interval of 6% for a 95% certainty.

2.7. pH Determination. pH values were measured with strips (Merk,Darmstadt, Germany) in the range of 0–6 and 5–10 pH units, with anerror of (0.5. Average value of two determinations was reported.

3. Results

A modification of alginate with �-cyclodextrin generatedthrough the addition of the CD moieties to the alginate carboxyl

742 Biomacromolecules, Vol. 9, No. 2, 2008 Santagapita et al.

groups was tested as invertase protective excipient. A schematicrepresentation of the synthesized �-CD biopolymer (�-CD-ethylenediamine-alginate) is shown in Figure 1. The bondingbetween alginate and the ethylendiamine-modified �-CD moi-eties was determined by 1H NMR spectroscopy, as shown inFigure 2. The appearance of multiple signals around 2.4–3.3ppm correspond to hydrogen nucleus in the �-CD rings andconfirm the presence of the branched �-CD units. In addition,the resonances associated to the methylene groups of theethylenediamine bridges are evident at higher fields (1.0–1.4ppm). The average �-CD content in the polymeric derivativewas estimated from the 1H NMR spectra by the integration ratiobetween the signals at 1.0–1.4 ppm (corresponding to the CH2

groups of the alkyl spacer) and those of the anomeric protonsof the carrier and of the �-CD moieties (4.3–5.4 ppm) byconsidering the number of protons involved in both groups of

signals. An average of 25 �-CD mol was estimated to beattached to each mol of modified alginate. The synthesizedpolymer had an average molecular weight (Mw, based on thetotal mass) of 1.8 × 105, as estimated by gel permeationchromatography. The polymer exhibited a high polydispersity,with Mw/Mn ) 3.2 (where Mn is the number molecular weightbased on the total number of mols). This ratio is an index ofhow widely distributed the range of molecular weights are inthe mixture (being Mw/Mn ) 1 in monodisperse polymers).

After drying, aliquots of the samples were resuspended andthe measured pH values were between 6 and 7 for all thesystems. Taking into account that the isoelectric point ofinvertase is lower than 4.5,40,41 it was assumed that the enzymewas negatively charged in the samples.

The invertase activity recovered after freeze-drying in theselected biopolymers and in their combined matrices withtrehalose is shown in Figure 3. During the drying, the enzymewas best protected by the trehalose matrix, while the polymersor their mixtures with trehalose were not as good as trehalose(T) to protect the enzyme. More than 80% of enzymatic activitywas recovered in alginate and dextran (with and withouttrehalose) and in �-CD+T (combined system of �-cyclodextrinwith trehalose) formulations. On the other hand, �-CD-A (with

Figure 1. �-CD-ethylenediamine-alginate (�-CD-A) structure representation. For synthesis details, see Section 2.1.

Figure 2. 1H-NMR spectrum for alginate (upper) and �-CD-A (lower)polymers. Both spectra are at the same scale. Water peak wasdeleted in order to clarify the spectra.

Figure 3. Activity of invertase in the selected biopolymers and in theircombined matrices with trehalose after freeze-drying in relation tothe activity of the enzyme without any additive. The bars representthe standard deviation.

Biopolymer Structure/Function and Enzyme Stability Biomacromolecules, Vol. 9, No. 2, 2008 743

and without trehalose) and �-CD systems showed an activity35–50% lower than the systems containing trehalose as the onlyexcipient.

Figure 4 shows that, although the sugar trehalose was aneffective protectant of the enzyme in systems exposed to RVP22% and heated at 87 °C, this effect was lost at RVP 43%.This behavior was related to trehalose crystallization.20–22,42,43

In the rehumidified systems at RVP 43%, the mobility and theamount of water were enough to allow trehalose dihydratecrystallization and the enzyme was rapidly inactivated. Amor-phous trehalose humidified to RVP 22% will not crystallize dueto the lack of water, and the enzyme was more protected.

Table 1 shows the remaining activity (RA) of invertase indifferent biopolymers or their mixtures with trehalose exposedto RVP 22% during one week and then incubated at 87 °C for25 h. In the combined systems, a synergistic stabilizing effectwas observed, with the exception of alginate containing matrices.

Figure 5 shows the remaining activity of the enzyme in thebiopolymer matrices and in the combined biopolymers/trehalosesystems exposed to RVP 43% as a function of heating time at87 °C. The best protective systems at RVP 43% were D+T(combined system of dextran with trehalose), �-CD+T, and�-CD-A+T (combined system of �-cyclodextrin-branched al-ginate with trehalose), followed by �-CD-A. Although �-CDcould interact with hydrophobic groups of the enzyme, prevent-ing denaturation-derived changes, it was not efficient as aprotectant (Figure 5a), probably due to its low solubility in water(1.8 g/100 mL), which precludes an adequate protein-polymerinteraction. The driving force of the guest-�-CD complexformation is the substitution of the high-enthalpy water mol-ecules inside the �-CD cavity by appropriate guest molecules,less polar than water, to occupy the relatively hydrophobic�-cyclodextrin cavity. The guest molecule and the �-cyclodex-

trin are usually dispersed in water and stirred to equilibriumfor several hours to favor inclusion complex formation.44

However, for labile compounds encapsulation, this procedurecould affect biomolecule stability, being inappropriate for oursystems. Through the addition of trehalose (system �-CD+T)or by its covalent modification with alginate (systems �-CD-Aand �-CD-A+T), �-CD becomes a suitable matrix to stabilizeinvertase. Both the addition of trehalose and/or the covalentmodification of �-CD seem to favor the interaction of �-CDwith hydrophobic groups of the enzyme, probably by increasingits water solubility. In fact, it was observed that the systems�-CD+T, �-CD-A, and �-CD-A+T were completely clear,indicating that solubilization had occurred. Alginate (system A)was a poor protectant of invertase, even in its mixture withtrehalose (A+T), as shown in Figure 5a,b. Enzyme stability canbe seriously affected by electrostatic interactions.45–47 Thus, theanionic characteristic of alginate probably affects the activeenzyme conformation. The physical properties of the matricesand their tendency to crystallize were studied in order to explainthe results on remaining invertase activity observed in Figure5. Differential scanning calorimetry (DSC) scans for selectedfreeze-dried samples exposed to RVP 43% are shown in Figure6. Table 2 shows the glass-transition temperatures and ∆Cp

value, degree of trehalose crystallization, and water contentvalues obtained for the freeze-dried samples exposed at 22 and43% RVP conditions. All systems were essentially amorphousat RVP 22%, confirmed by the absence of the meltingendothermal peaks in the DSC thermograms (data not shown).Both alginate and dextran presented higher Tg values thantrehalose, and �-CD-A showed Tg values similar to �-CD atlow RVP. The decrease of Tg value as increasing water contentwas less pronounced in �-CD-A systems than in the rest of thesystems (Table 2), reflecting an effect of alginate modification.

Figure 4. Remaining activity of the enzyme invertase in trehalosefreeze-dried systems exposed at RVP 22% and 43% as a functionof incubation time at 87 °C.

Table 1. Remaining activity (RA) for systems exposed at RVP22% during one week and then incubated at 87 °C for 25 ha

RA (%)

polymer without trehalose with trehalose

�-CD 6.2 71.2D 41.2 80.1�-CD-A 37.8 67.2A 47.1 42.7none 63.5

a �-CD: �-cyclodextrin; D: dextran; �-CD-A: �-cyclodextrin-ethylenedi-amine-alginate; A: alginate.

Figure 5. Remaining activity of the enzyme invertase in the freeze-dried systems exposed at RVP 43% as a function of heating time at87 °C. (a) Polymer systems without trehalose; (b) combinedtrehalose-biopolymer systems.


In trehalose systems, the amount of adsorbed water at RVP 43%was sufficient for complete trehalose crystallization (the amountof water to form trehalose dihydrate is 10.5% in dry basis).While the pure trehalose system had crystallized to a high extentduring the humidification step at RVP 43% (the degree ofcrystallization was 60% and increased up to 80% during thethermal treatment), sugar crystallization was delayed/inhibitedin the blends containing any of the studied polymers (Figure 6and Table 2). Thus, the synergistic stabilizing effect of thecombined systems �-CD+T, �-CD-A+T, and D+T could be,at least in part, related to the delay/inhibition of trehalosecrystallization, although molecular interactions played a sig-nificant role. �-CD-A has shown to be an appropriate additivefor dehydrated formulations of invertase, allowing protectionof the enzyme in conditions at which trehalose crystallizes(Figure 5a,b).

Parts a and b of Figure 7 show the atomic force microscopy(AFM) images of films obtained by dip coating of the modifiedpolymer, �-CD-A, and of alginate. The difference in roughnessin both a and b images indicates that supramolecular structuresformed in the unmodified alginate are different from those inthe modified �-CD-A. Alginate showed aggregates presentingtopographical patterns of bigger size and height than those of�-CD-A. The CD-moieties incorporated to the alginate backbonein �-CD-A may preclude certain repulsive interactions betweenalginate chains of the same charge.

4. Discussion

Although both alginate and dextran were good physicalstabilizers due to their high Tg values (compared to trehalose),their action as a single-additive protectant was only moderateto poor. It is to be noted that electrostatic forces determineprotein interactions with biopolymers,45–47 which may positivelyor negatively influence protein stability. Alginate is a polyan-ionic polymer (at pH used in the present work), and itstridimentional structure exposes negative residues to the surfaceof the enzyme in the dehydrated system, probably destabilizingthe native structure of the protein at the interface with thepolymer. Dextran is a neutral polymer, and the enzyme stability

is not affected by electrostatic interactions, but in this case, thestabilization effect was moderate. �-CD is also neutral, and itcan interact with hydrophobic groups of the enzyme preventingunfolding, but it was not efficient as a protectant. The covalentcombination of �-CD with alginate in the new polymer enhancedthe enzyme stability, possibly due to a combination of factors.�-CD-A lacks of some unfavorable electrostatic interactionspresent in alginate (various carboxylic groups of alginate areblocked by �-CD units). It is expected that �-CD attached tothe polymer has higher accessibility to hydrophobic residuesof the protein than single �-CD molecules due to the lowsolubility in water of the latter. Then, the complex between thecyclodextrin ring and the hydrophobic aminoacids of the proteincan be formed in a higher extent, helping to prevent proteinunfolding.4,5,10,48 The �-CD-A polymer also provides goodphysical stability, given by a relatively high Tg value, as shownin Table 2. Besides, the water effect on decreasing Tg valueswas less pronounced in �-CD-A system, probably due tochanges of water-biopolymer interactions promoted by thechemical modification with CD moieties, which are lesshygroscopic than carboxylic groups of alginate.

It is also to be noted that in very diluted liquid systemspreviously analyzed (cyclodextrin polysucrose or dextranpolymers)10,29 or in present supercooled systems, the cyclodex-trin alone or the unmodified polymer were not able to performprotective effect on the analyzed enzymes, and in all cases, theintroduction of CD moieties in a polymer chain improved theenzyme stability.

While trehalose protective activity is lost at 43% RVP, themodified �-CD-A, as a single additive, exhibited enzymeremaining activity values higher than 60% after 25 h of thermaltreatment at 87 °C. Among the binary additive systems, trehalosecombined with �-cyclodextrin (�-CD+T), with dextran (D+T),or with the new polymer (�-CD-A+T) offered the best stabilityto the enzyme. In our combined systems, trehalose wasresponsible for the higher protection observed at 22% and 43%RVP, because the single-additive systems containing polymers�-CD or D did not confer adequate stability to the enzyme. Itis also interesting to note that, in the A+T system, thedestabilization effect of alginate predominates over the protectiveeffect of trehalose, possibly due to unfavorable electrostaticprotein-biopolymer interactions. It has been shown before thata high glass transition for the excipient is not a critical factorin enzyme stabilization during drying49 and that the mobilityof the protein in a glass is not necessarily well measured by thedifference between the glass transition temperature of the pureexcipient and the sample temperature. The effect of polymersin the mixtures with trehalose was the delay/inhibition of thesugar crystallization. In noncrystallized systems (RVP 22%),the synergistic stabilizing effect observed for some combinedsystems (Table 1) could be attributed to physical stabilityprovided by the polymer and trehalose acting as filler and totrehalose-enzyme hydrogen bond interactions.

The molecular packing of the excipient could be anotherparameter to be considered: alginate has a linear structure, andthe repulsions between molecules could be responsible for thelarger aggregated structures shown in Figure 7, with a probablylooser molecular packing in the freeze-dried matrix than thoseof �-CD-A. The latter has a branched structure in which lessrepulsive interactions due to the chemical modification allow abetter packing to protect the enzyme. The way in which theamorphous matrices are packed determines the presence of voidsor defects and may affect their protective properties. Hemmingaet al.50 observed that the rotational mobility of a paramagnetic

Figure 6. Differential scanning calorimetry (DSC) thermograms forselected freeze-dried samples exposed to RVP 43% during one week.Glass-transition temperature (Tg) and trehalose melting are indicated.


probe increased when the number of maltose units increased inmaltodextrin polymers. Matrices formed by small molecules(i.e., disaccharide) are more densely packed than high-molar-mass saccharides, thus they are more effective to restrict themolecular mobility of the embedded compounds than matricesformed by compounds with a low degree of molecular packing(like cyclodextrin or starch). Trehalose forms hydrogen bondswith biomolecules,51 and the high density molecular packingof the matrix determines its low free volume.50 Mazzobre etal.52 showed that, in anhydrous conditions, the remaining activityof lactase in the presence of various additives displays amaximum corresponding to maltodextrins of intermediate mo-lecular weight. This fact reflects an interesting compromisebetween the benefits of having high Tg values and of themolecular packing structure, concluding that the stability ofenzymes in dried matrices was not only related to supramo-lecular aspects or to the molecular mobility of the matrix, butit was also dependent on other factors such as chemicalreactivity, molar mass, conformation, or molecular structure ofthe matrix.52 �-CD-A combines good physical characteristics

(high Tg value) and adequate molecular matrix packing, whichprevent protein unfolding and could also favor �-CD-enzymeinteractions.

This paper shows that a relatively simple covalent combina-tion of two biopolymers significantly affects their functionalitiesand, consequently, their interactions with proteins, modifyingenzyme stability patterns. The covalently linked alginate with�-CD has shown to protect dehydrated formulations of invertase,avoiding its unfavorable electrostatic interactions with alginate.This modified polymer was effective even at conditions at whichone of the most accepted excipients for protein formulations(trehalose) lost its protective effect. On the other side, themodification couples �-CD moieties to a widely used biopoly-mer, increasing or improving both biopolymers’ potentialapplications.

Acknowledgment. We acknowledge Julian Gelman-Con-stantin for assistance in AFM technique and the financial supportof SECYT-CITMA (bilateral project CU/PA04-EVI/037),CONICET (PIP 5977), UBACyT X-226, and ANPCYT (PICT32916, 20545, and 20680).

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Table 2. Glass-Transition Temperature (Tg), Change in Heat Capacity at Tg (∆Cp), Degree of Trehalose Crystallization (�), and WaterContent Values (as percent of dry basis, % db) Obtained for Samples Exposed at 22 or 43% RVP during 1 Weeka

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system water content (% db) Tg (°C), onset ∆Cp (J/gK) water content (% db) Tg (°C), onset ∆Cp (J/gK) � (%)

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a T: trehalose; �-CD: �-cyclodextrin; D: dextran; A: alginate; �-CD-A: �-cyclodextrin-ethylenediamine-alginate. � ) 100 ∆Hsample/∆Hpure trehalose.

Figure 7. Atomic force microscopy images of films obtained by dip-coating: (a) alginate and (b) �-CD-A. Both images correspond to asurface of 1 µm × 1 µm. Left pictures correspond to height data (scaleindicated in each picture); right pictures correspond to deflection data(maximum of 0.5 nm for both pictures).


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Structure/Function Relationships of Several Biopolymers...

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