Tailoring the Network Properties of Ca2+ Crosslinked Aloe veraPolysaccharide Hydrogels for in Situ Release of Therapeutic

Agents

Shawn D. McConaughy, Stacey E. Kirkland, Nicolas J. Treat, Paul A. Stroud,| andCharles L. McCormick*,,

Department of Polymer Science, Department of Chemistry and Biochemistry, The University of SouthernMississippi, Hattiesburg, Mississippi 39406, DelSite Biotechnologies, Irving, Texas 75038

Received July 29, 2008; Revised Manuscript Received September 10, 2008

Properties of Aloe Vera galacturonate hydrogels formed via Ca2+ crosslinking have been studied in regard to keyparameters influencing gel formation including molecular weight, ionic strength, and molar ratio of Ca2+ to COO-

functionality. Dynamic oscillatory rheology and pulsed field gradient NMR (PFG-NMR) studies have beenconducted on hydrogels formed at specified Ca2+ concentrations in the presence and absence of Na+ and K+ ionsin order to assess the feasibility of in situ gelation for controlled delivery of therapeutics. Aqueous Ca2+

concentrations similar to those present in nasal and subcutaneous fluids induce the formation of elastic Aloe Verapolysaccharide (AvP) hydrogel networks. By altering the ratio of Ca2+ to COO- functionality, networks may betailored to provide elastic modulus (G) values between 20 and 20000 Pa. The Aloe Vera polysaccharide exhibitstime-dependent phase separation in the presence of monovalent electrolytes. Thus the relative rates of calciuminduced gelation and phase separation become major considerations when designing a system for in situ deliveryapplications where both monovalent (Na+, K+) and divalent (Ca2+) ions are present. PFG-NMR and fluorescencemicroscopy confirm that distinctly different morphologies are present in gels formed in the presence and absenceof 0.15 M NaCl. Curve fitting of theoretical models to experimental release profiles of fluorescein labeled dextransindicate diffusion rates are related to hydrogel morphology. These studies suggest that for efficient in situ releaseof therapeutic agents, polymer concentrations should be maintained above the critical entanglement concentration(Ce, 0.60 wt %) when [Ca

2+]/[COO-] ratios are less than 1. Additionally, the monovalent electrolyte concentrationin AvP solutions should not exceed 0.10 M prior to Ca2+ crosslinking.

1. Introduction

Galacturonates, commonly termed pectins, are naturallyoccurring polyelectrolytes with characteristics especially ame-nable to controlled release applications.1 These polysaccharides(Figure 1) have also been widely used in the food industry2,3

and are composed primarily of (1f 4)R-D-galacturonic acid(GalA) repeat units and contain regions that include (1f2)linked rhamnose residues that act as branch points for neutralsugars. The carboxyl units along the backbone provide saltresponsiveness and allow formation of hydrogel networks whendivalent ions such as Ca2+ are introduced (Scheme 1).4-7 Recentstudies regarding Ca2+ binding to pectins and closely relatedalginates8,9 have revealed differences in the two systems,suggesting that Ca2+ binding in pectins occurs in a two stageprocess10 and results in a shifted egg box structure.11 Theoral delivery of small molecules including colon-specificdrugs12,13 from pectin hydrogels formed by Ca2+ crosslinkinghave been extensively studied and excellent reviews of thesubject are available in the literature.1,14-16 However, effectivemethods by which networks may be tailored for controlleddelivery of macromolecular species such as protein therapeuticsremain undeveloped.17-19 For example, oral delivery of proteins

requires efficient transportation across the gastrointestinal (GI)tract membrane and limiting enzymatic and hydrolytic proteindegradation.16

Alternative routes that circumvent some of the aforementionedissues involve protein delivery via subcutaneous injection orintroduction through the nasal cavity by adsorption of proteinsat the epithelial surface.20-22 The latter route results in directentry of the therapeutic agent into systemic circulation.18,20 Thepresence of Ca2+ in mucosal and subcutaneous fluids providesa natural source for in situ gelation of carboxylated polymers.Given the low concentrations (3-5 mM) of Ca2+ present inmucosal23 and subcutaneous fluids,24 suitable macromoleculesmust have a large number of carboxyl functional groupsavailable for crosslinking. Previous research in our laboratories25

has shown that a polysaccharide extracted from the Aloe Veraplant has a high galacturonic acid content and low degree ofmethyl ester substitution that allows for facile gel formation inthe presence of Ca2+ at relatively low concentrations. Interest-ingly, the Aloe Vera polysaccharide exhibits phase separationover time at ionic strengths similar to those of biological fluids.Thus, the relative rates of calcium induced gelation and phaseseparation become major considerations when designing asystem for in situ delivery applications where both monovalent(Na+, K+) and divalent (Ca2+) ions are present.

In this research, we report the gelation behavior and matrixcharacteristics of Ca2+ crosslinked AvP hydrogels. Additionally,we investigate the effects of inducing phase separation byaddition of monovalent electrolytes prior to Ca2+-inducedgelation. The matrix characteristics of AvP hydrogels formed

Paper 135 in a series entitled Water Soluble Polymers.* To whom correspondence should be addressed. E-mail: charles.

mccormick@usm.edu. Department of Polymer Science, The University of Southern Mississippi. Department of Chemistry and Biochemistry, The University of Southern

Mississippi.| DelSite Biotechnologies.

Biomacromolecules 2008, 9, 32773287 3277

10.1021/bm8008457 CCC: $40.75 2008 American Chemical SocietyPublished on Web 10/21/2008

in solutions at the ionic strengths and molar [Ca2+]/[COO-]ratios of physiological fluids have been determined based onviscoelastic behavior and PFG-NMR studies of water diffusion.To establish relationships between AvP network properties andthe diffusion behavior of macromolecules through the gel, therelease profiles of fluorescein labeled dextrans have beenmeasured as a model for therapeutic proteins. The results ofthis study serve as a basis for establishing guidelines formonovalent salt and polymer concentrations, as well as [Ca2+]/[COO-] ratios, appropriate for in situ AvP crosslinking and thecontrolled release of therapeutic agents in nasal or subcutaneousenvironments.

2. Materials and Methods

2.1. Materials. Aloe Vera polysaccharide trademarked as GelSitepolymer was donated by DelSite Biotechnologies (Irving, TX). Thepectin was isolated by extraction with ethylene diamine tetra-aceticacid (EDTA) from the rind of Aloe Vera L. The primary sample usedin this study, AvP2, has a Mw of 435 kDa and is composed of 95%galacturonic acid (GalA) residues of which 5% are in the methyl esterform. Details of the chemical composition of AvP2 and 2 other samplesutilized in this study are included in Table 1. Further details concerningthe chemical composition and dilute solution properties of AvP havebeen previously described.25

2.2. Solution Studies. Turbidity. To determine the stability of AvPin aqueous solutions containing monovalent salts, turbidity wasmonitored via measurement of absorbance at 410 nm and 3-dimensionaldiagrams of absorbance as a function of polymer and salt concentrationwere constructed. Stock polymer (4 mg/mL) and NaCl (0.40 M)solutions were prepared in deionized water containing 5 ppm sodium

azide. Samples were prepared at appropriate polymer/ionic strengthcombinations in a 96-well plate using a Biomek FX liquid handlersystem. Analysis was conducted using a Tecan Saphire dual fluores-cence and UV-vis detector. Turbidity at 25 C was measured at 1 htime intervals. Phase diagrams were constructed from approximately96 data points utilizing the mesh feature of DPlot software version2.1.3.8. Results were confirmed via visual monitoring of separatesolutions prepared in 15 mL scintillation vials.

2.3. Hydrogel Characterization. Sample Preparation. AvP wasdissolved in DI water containing 5 ppm sodium azide and stirredovernight. The resulting solutions were then diluted with an appropriateNaCl solution to reach the desired polymer/NaCl concentrations andstirred for at least 1 h. When examining the effect of phase behavioron the ability of AvP to form gels in the presence of divalent ions,polymer/NaCl solutions were mixed for the desired time (2-24 h) priorto crosslinking with Ca2+ ions. The pH of all solutions was ap-proximately 6.7, a value at which AvP is considered to be fully ionized(pKa 3.4). AvP hydrogels were formed by crosslinking with CaCl2in a custom designed mold. The mold consisted of an upper and lowerreservoir, in which 5 mL of AvP solution (1-8 mg/mL) was placed inthe lower reservoir. This reservoir was then covered with 6-8 kDaMW cutoff dialysis tubing (Spectra/Por), and 5 mL of the desired CaCl2solution (3-50 mM) was gently pipetted into the upper reservoir.Diffusion of Ca2+ from the upper to lower reservoir initiated hydrogelformation. Experiments were conducted to determine the CaCl2exposure time necessary to reach equilibrium gel strength (discussedin Section 3.2). Unless otherwise stated, hydrogels discussed in thispublication were given 24 h to ensure that equilibrium conditions werereached.

Dynamic Rheology. Oscillatory dynamic rheological experimentswere conducted with an ARES-G2 stress controlled rheometer equipped

Figure 1. General structure of the Aloe vera pectin including galacturonic acid units with methyl esters (black filled square), galacturonic acidsodium salt form (red filled circle), rhamnose (green filled upward-pointing triangle), and neutral sugar branches (R).

Scheme 1. Idealized Representation of Calcium Induced Crosslinking

Table 1. Chemical Composition and physical parameters of Aloe vera Polysaccharide (AvP)

pectin Mwa (kDa) PDI Rga (nm) GalAb Rhab Manb Glab Arab Glub Fucb Xylb DMc DMd

AvP1 523 1.62 115 95.8 0.46 2.02 1.04 0.31 0.19 0.16 0.06 6 5.3AvP2 435 1.58 103 94.5 0.49 2.4 1.39 0.53 0.41 0.17 0.12 3 5.3AvP5e 200 NDf NDf 97.1 0.33 1.37 0.69 0.15 0.16 0.14 0.08 NDf 4.7

a Obtained from SEC-MALLS. b Listed as percent of total carbohydrates. c Listed as mole percent of GalA. d Listed as mole percent of GalA as determinedby H1 NMR. e Sample prepared via hydrolysis. f Not determined.

3278 Biomacromolecules, Vol. 9, No. 11, 2008 McConaughy et al.

with a 40 mm crosshatched parallel plate. A stress of 0.50 Pa wasutilized in experiments performed as a function of frequency (0.10-100rad/s). Separate experiments were performed as a function of stress(0.01-10 Pa) at 0.5, 10, and 100 rad/s in order to ensure that a stressof 0.50 Pa was within the linear viscoelastic regime of AvP hydrogels.All gels were tested at 25 C after compression to a normal force of0.30 N. Samples were tested in triplicate. Calculated values of standarddeviation were typically around 5% and did not exceed 10%.

Pulsed Field Gradient Nuclear Magnetic Resonance. AvP hydrogelswere prepared directly in NMR tubes. AvP solution (30 L, 0.20 or0.60 wt %) was placed in a 5 mm NMR tube to which 30 L of theappropriate CaCl2 solution was added. Gels were given 24 h to reachequilibrium prior to NMR analysis. Sample volumes were kept at 60L in order to optimize signal-to-noise ratios during the pulsed fieldgradient (PFG) experiments. All spectra were obtained with a VarianUnity Inova 500 MHz spectrometer using a standard 5 mm 2 channelprobe equipped with gradients. The standard Stejskal-Tanner se-quence26 (acquisition time of 0.5 s, a recycle delay of 5 s, and gradientpulses of 0.8-1.0 ms) was utilized in the PFG-NMR experiments todetermine the time-dependent diffusion coefficient of water (Dapp). Theself-diffusion coefficient of water was determined from the negativeslope of a log-attenuation plot (log versus 2g22( - /3)), where is the echo attenuation, is the proton gyromagnetic ratio, is thewidth of the gradient pulse, g is the magnitude of the applied fieldgradient, and is the total diffusion time. The total diffusion timewas varied from 20 to 500 ms, and the gradient amplitude ranged from20 to 80 G/cm to ensure the signal was attenuated 80%. The spectralwidth was 50 kHz, and the number of scans for each spectrum rangedfrom 8-32. Exponential line broadening was applied prior to Fouriertransformation of the FIDs. Gradient calibration was performed usinga deionized water standard prior to data collection.

Microscopy. Bright field and flouresence images were obtained witha Nikon Eclipse 80i microscope, and images were processed utilizingNIS-elements f software. Thin hydrogel samples were prepared directlyon cleaned glass slides. Samples were stained with 0.10 wt % rutheniumred, which has been shown to effectively stain pectins.27

2.4. Release Studies. Materials and Sample Preparation. Fluores-cein labeled 4 kDa and 500 kDa Mw dextrans (Dex4, Dex500) werepurchased from Sigma-Aldrich and utilized as model compounds incontrolled release experiments. To minimize photobleaching, releaseexperiments involving fluorescein labeled dextrans (FITC-dextrans)were performed in a dark room under red light. Stock AvP and FITC-dextran solutions were dissolved overnight and combined to yield fourstock solutions at AvP concentrations of 6 and 2 mg/mL, a FITC-dextran concentration of 0.10 mg/mL, and a NaCl concentration of0.05 M. All experiments were conducted in triplicate (standarddeviations averaged 5% and were used to create error bars). Sampleswere prepared in 1.5 mL microcentrifuge tubes and contained 0.5 mLof AvP/FITC-dextran dissolved in 0.05 M NaCl. To create a consistentinterface between the AvP/FITC-dextran solution and the releasemedium, a Teflon grid with a macroscopic grid opening (1 mm 0.635mm) (McMaster-Carr) was placed on top of the AvP/FITC-dextransolution. Next, 1 mL of a simulated nasal fluid (SNF) composed of 10mM Tris, 0.15 M NaCl, 0.04 M KCl, and 5 mM CaCl2 was added.Subsequent diffusion of Ca2+ into the AvP/FITC-dextran solutioninitiated crosslinking; release of the FITC-dextran into the SNF solutionwas then monitored. Then 500 L aliquots were taken at various timeintervals, and the release medium was replaced with fresh SNF.

Fluorescence Detection. The fluorescence emission (510-600 nm)of FITC-dextrans present in aliquots was measured at an excitationwavelength of 495 nm on a Photon Technology International spec-trometer. Calibration curves were constructed for both FITC-dextranas well as FITC-dextran/polymer solutions, and separate experimentswere conducted in order to ensure that sink conditions were maintainedin the release medium throughout the experiment. After 4 days, therelease experiment was halted. To determine the amount of free dextranremaining in the gels, gels were suspended in fresh SNF within 1.5

mL microcentrifuge tubes and centrifuged for 2 min at 1000 rpm. Thesolutions were collected, and fluorescence emission was measured. Todetermine if FITC-dextran was permanently entrapped within thecalcium hydrogels, the gels were dissolved in a 0.5 M EDTA solutionovernight, and fluorescence of the resulting solutions was measured.

Model Analysis. Curve fitting was performed using a nonlinear curvefitting tool from Origin software (version 7.0383). Additional analysisof the squared sum of residuals (SSR) between experimental data andtheoretical data was conducted in order to determine the goodness offit for each diffusion model. SSR values were substituted into the Akaikeinformation criterion (AIC) defined by eq 1:

AIC)N(ln SSR)+ 2p (1)N accounts for the number of data points being compared, and prepresents the number of variables used in model fitting. The best fit isrepresented by the lowest value of AIC.28

3. Results and Discussion

3.1. Structure/Solution Properties of AvP. Recently, wereported in detail the chemical composition (Table 1) andaqueous solution behavior of a galacturonate polysaccharidederived from Aloe Vera.25 Our studies demonstrated that whencompared to traditional pectins derived from citrus sources, AvP(Figure 1) galacturonic acid repeat units have a low degree ofmethylester substitution (5%). Over a range of molecularweights (200-500 kDa) and in the presence of monovalentelectrolytes, AvP retains a solvated, extended conformation,which allows facile Ca2+-induced hydrogel formation (Scheme1).

3.2. Hydrogel Preparation and Characterization. To de-termine network characteristics, hydrogels were prepared asdetailed in the experimental section by introducing calciumchloride solutions of desired concentration into a reservoircontaining AvP solutions. A membrane was placed on top ofthe AvP solution to ensure uniform diffusion as Ca2+-inducedgelation occurred. This procedure not only allows experimentalcontrol of reaction parameters including polymer concentration,ionic strength, and [Ca2+]/[COO-] ratios but also mimics in apractical manner in situ gelation for therapeutic deliveryapplications.

3.2.1. Viscoelastic BehaVior of AVP Networks. MolecularWeight and Chemical Composition. AvP samples with molecularweights of 200, 435, and 500 kDa were dissolved at 0.10 wt %and crosslinked via introduction of 5 mM CaCl2. After allowinga 24 h reaction time, AvP solutions formed clear hydrogels thatwere easily transferred from the mold and studied by dynamicrheometry. As shown in Figure 2, the elastic moduli (G) of all

Figure 2. Elastic (filled symbols) and viscous (open symbols) modulusdata as a function of frequency a series of 0.10 wt % AvP hydrogelsformed from AvP samples of molecular weights 523 (black filledsquare), 435 (red filled circle), 200 (green filled upward-pointingtriangle) kDa 24 h after introduction of 5 mM CaCl2.

Ca2+ Crosslinked Aloe vera Polysaccharide Hydrogels Biomacromolecules, Vol. 9, No. 11, 2008 3279

samples were much greater than the viscous moduli (G) andwere essentially linear as a function of frequency. The variationin chemical composition between AvP samples (Table 1) issmall and does not significantly affect G values after Ca2+crosslinking. Additionally, G appears to be independent of AvPmolecular weight over the 200-500 kDa range studied here. Itshould be noted that this is not the case for low molecular weightpectins. Previous studies have been conducted that examineelastic modulus-molecular weight relationships of polysaccha-ride hydrogels.29 In studies utilizing 6, 22, and 66 kDa pectins,Durand et al.30 have shown that low molecular weight speciesare less effective at forming elastically active networks. Ap-parently, their existence as rigid rods in solution hinders theformation of elastically active junctions in the hydrogels.Because the molecular weights of all AvP samples studied inour work are well above the rod limit,25 no variation in hydrogelelastic modulus is evident. Given the structural regularity ofthe AvP polymers and molecular weight independence of gelproperties, AvP2 (Table 1) was chosen for the remainder ofthe studies reported here.

Rate of Ca2+ Crosslinking. Dynamic oscillatory measure-ments conducted over a wide frequency range illustrate theexpected increase in values of G and G with increasinggelation time for AvP2 at concentrations of 0.20 and 0.60 wt% in 5 mM CaCl2. G is much greater than G and both arelinear as a function of frequency, as illustrated for 0.20 wt %hydrogels in the Supporting Information, Figure S1. Examina-tion of the values of G and G as a function of time revealsthat, under these conditions, most gelation occurs within thefirst six hours, after which asymptotic values of G and G arereached (Figure 3). These results are in agreement with studiesconducted by Silva et al.31 To ensure that equilibrium had beenreached, oscillatory rheology studies were conducted on AvPgels 24 h after introduction of Ca2+.

Polymer Concentration. The network characteristics ofbiopolymer gels are often heavily dependent on the concentra-tion of polymer present in the system.32-34 In the case of pectinhydrogels, polymer concentration has been shown to be a keyfactor affecting the final pectin network characteristics.35,36

AvP2 solutions at concentrations ranging from 0.10 to 0.80 wt% were crosslinked with 3, 5, 15, 35, and 50 mM CaCl2.Oscillatory rheology conducted as a function of frequencyprovides values of G, G, and tan (Table 2) that can beutilized as a diagnostic of gel rigidity.37 AvP hydrogels exhibitstrong gel behavior (tan

nasal and subcutaneous fluids, which is approximately 5 mM.Two samples (highlighted data points in Figure 5) havingsufficiently high moduli for hydrogel integrity were chosen forthe diffusion and controlled release studies addressed insubsequent sections of this manuscript. It is important to notethat, although prepared at substantially different polymerconcentrations (0.20 vs 0.60 wt %), the experimentally measuredvalues of G are similar (500 Pa) for [Ca2+]/[COO-] ratiosof 0.5 and 0.2, respectively.

Addition of Simple Electrolytes. Electrolyte addition to anionicpolysaccharides lowers hydrodynamic volume in aqueous solu-tion by effective charge screening and by reduction of polymersolvent interactions. Although the conformationally stiff AvPis less prone to viscosity loss when compared to flexiblepolyelectrolytes such as poly(sodium acrylate), addition ofelectrolytes such as NaCl reduces hydrodynamic volume. ForAvP2, a 17% decrease in intrinsic viscosity was observed asNaCl concentration was increased from 0.05 to 0.20 M.25 Theseexperimentally determined effects on conformation and solvation

are expected to be manifested in properties of the crosslinkedgel matrices as well.

Another critical issue arising from changes in solvation fromadded electrolytes is the possibility of phase separation andaggregation. While conducting previous intrinsic viscositystudies on AvPs, we observed phase separation in dilutesolutions at ionic strengths above 0.15 M.25 A closer examina-tion reveals gradual association of AvP chains that becomesnoticeable at extended time. For example, initially linearHuggins-Kraemer plots (2 h) become nonlinear after 24 h(Supporting Information, Figure S4). Further evidence wasobtained from potential and dynamic light scattering studies.At low ionic strength, the potential is -80 mV and thesolutions are stable, however,as the ionic strength is increasedto 0.10 M, the potential approaches -30 mV.25

Turbidimetric experiments were conducted on AvP2 solutionsfor a wide range of polymer concentrations and ionic strengthsas detailed in the Materials and Methods Section. Polymerconcentrations were chosen between 0 and 0.20 wt %, and theionic strength was assumed to be that of the NaCl solution.Turbidity measurements, specifically the three-dimensional plotsshown in Figure 6a,b, confirm that the extent of phase separation

Table 2. Experimentally Determined Elastic (G) and Viscous (G) Moduli Reported at a Frequency of 6.2 rad/s for AvP HydrogelsCrosslinked by Ca2+

0.10 wt % AvP 0.20 wt % AvP

[Ca2+] (mM) [Ca2+] [COO-] G (Pa) G (Pa) tan G/G crossover

(rad/s) [Ca2+] [COO-] G (Pa) G (Pa) tan G/G crossover

(rad/s)

3 0.62 29 24 1.09 2.1 0.31 17 11 0.80 3.95 1.03 190 22 0.11 NAa 0.52 500 54 0.11 1.1

15 3.08 460 45 0.10 NA 1.54 1500 190 0.13 NA25 5.14 540 58 0.11 NA 2.35 1500 200 0.13 NA35 7.19 490 57 0.12 NA 3.60 1600 210 0.13 NA50 10.27 550 64 0.12 NA 5.14 1700 240 0.14 NA

0.60wt%AvP 0.80wt%AvP

[Ca2+] (mM) [Ca2+] [COO-] G (Pa) G (Pa) tan G/G crossover

(rad/s) [Ca2+] [COO-] G (Pa) G (Pa) tan G/G crossover

(rad/s)

3 0.10 120 21 0.17 NA 0.08 1300 440 0.34 NA5 0.17 560 88 0.16 NA 0.13 2400 570 0.28 NA

15 0.34 2500 370 0.15 NA 0.39 5200 1200 0.24 NA25 0.52 8500 1290 0.15 NA 0.64 16000 2800 0.18 NA35 0.86 9800 1300 0.14 NA 0.90 24000 4100 0.17 NA50 1.71 12000 1600 0.13 NA 1.28 26000 3500 0.13 NA

a NA: Not applicable, G and G were linear as a function of frequency and no crossover was observed.

Figure 4. Elastic modulus plotted against AvP2 concentration ofhydrogels formed at CaCl2 concentrations of 3 (black filled squares),15 (red filled circles), 35 (green filled upward-pointing triangles) and50 (blue filled downward-pointing triangles) mM. The y-axis of theinset is scaled to depict the 3 mM CaCl2 series only and draw attentionto the dramatic increase in G evident for hydrogels formed fromconcentrated solutions (>0.60 wt %).

Figure 5. Elastic modulus (Pa) plotted as a function of the molar ratio[Ca2+]/[COO-] for AvP2 calcium gels formed at concentrations of 0.10(black filled squares), 0.20 (red solid circles), 0.40 (green filledupward-pointing triangles), 0.60 (blue filled downward-pointing tri-angles), 0.80 (green filled diamonds) wt % polymer. Shaded areacorresponds to Ca2+ concentrations found in nasal fluids. The boxedarea indicates the region from which hydrogels were evaluated inrelease studies described in Section 3.3 of the discussion.

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is time-dependent and related to polymer concentration and ionicstrength. After 2 h, the extent of phase separation observed insolutions containing greater than 0.10 wt % AvP at ionicstrengths greater than 0.15 M is moderate. However, Figure 6bshows that phase separation is more prevalent after 24 h. Itshould be pointed out that, although phase separation can occurin the presence of NaCl, the time dependency for this processis slow relative to the rate of Ca2+-induced gelation (Figure 3).

On the basis of our studies and previous reports regardingthekineticcompetitionbetweenphaseseparationandgelation,46,50,51

we anticipated that sample history would affect networkproperties and therefore prepared hydrogels from two series of0.20 wt % AvP2 solutions with 0, 0.05, 0.10, 0.15, and 0.20 MNaCl aged for 2 and 24 h, respectively. These compositionsare indicated by the open circles in Figure 6a,b and include;homogeneous solutions (lowest turbidity), microphase separatedsolutions (moderate turbidity), and phase separated colloidaldispersions (highest turbidity). Elastic and viscous modulusprofiles as a function of frequency are recorded in Figure S5 ofthe Supporting Information.

Both the effects of ionic strength (NaCl concentration) ofthe AvP2 solutions and aging time prior to crosslinking can beascertained by examination of Figure 7. The single referencepoint on the left side of the plot represents the hydrogel modulusvalue of 1500 Pa after Ca2+ (35 mM) crosslinking of a 0.20 wt% solution of AvP2 in the absence of NaCl (0.00 M). Increasesin experimentally measured G values are observed reaching2300 Pa at 0.15 M NaCl before falling abruptly at higher ionic

strength to 600 Pa in the samples aged for 2 h prior tocrosslinking. Smaller but discernible increases in G are observedfor AvP2 samples aged for 24 h prior to crosslinking thatcontained 0.05 and 0.10 M NaCl. Because the modulus (Figure7) and turbidity (Figures 6a,b, data points indicated by circles)measurements are on the same samples, it appears that lowconcentrations of NaCl induce associations that are helpful tonetwork formation. Higher levels of association which occurwith increased aging and/or NaCl concentration result in totalphase separation and incomplete gelation.

To simulate Ca2+ crosslinking at the ionic strength of a nasalfluid, homogeneous aqueous solutions of 0.20 wt % AvP werecrosslinked via introduction of a solution containing 5 mMCaCl2, 0.15 M NaCl, and 0.04 M KCl.

23 When crosslinkedunder these conditions, aqueous AvP solutions form hydrogelswith an elastic modulus value of 1200 Pa, representing asignificant increase over the 500 Pa value obtained when AvPsolutions are crosslinked by 5 mM CaCl2 alone (Figure 8). Theformation of a strong hydrogel is consistent with the absenceof large scale phase separation, further supporting the conclusionthat the rate of Ca2+ crosslinking is fast relative to the rate ofphase separation. However, when simulated nasal fluids (SNF)are employed in the crosslinking reaction, an increase in Goccurs that is similar to that observed for moderate NaClconcentrations (Figure 7).

3.2.2. Diffusion Studies Via PFG-NMR. Information regardingpores within the viscoelastic hydrogel matrix can be gained fromdiffusion studies utilizing pulsed field gradient NMR (PFG-NMR). In PFG-NMR experiments, the apparent diffusion

Figure 6. Turbidity of AvP2 solutions as functions of NaCl (M) and polymer (wt %) concentration at 2 (a) and 24 (b) hours. Selected combinationsof salt and polymer concentration (blue filled circles) were used in Ca2+ induced gel formation studies in order to determine the effect of phasebehavior on hydrogel elastic modulus (Figure 7).

Figure 7. Elastic modulus of 0.20 wt % AvP2 hydrogels formed bycrosslinking with 35 mM CaCl2. Aqueous AvP2 solutions at thespecified NaCl concentration shown along the abscissa were agedfor 2 (black filled squares) and 24 (red filled circles) h prior to additionof CaCl2 solutions.

Figure 8. Elastic modulus of 0.20 wt % AvP2 hydrogels formed bycrosslinking with 5 mM CaCl2 and a simulated nasal fluid (SNF)containing 5 mM CaCl2, 0.15 M NaCl, and 0.04 M KCl.

3282 Biomacromolecules, Vol. 9, No. 11, 2008 McConaughy et al.

coefficient of water (Dapp) is monitored as a function of totaldiffusion time (). In restricted geometries (such as hydrogelmatrixes), Dapp is unhindered at short diffusion times and equalsthe diffusion coefficient of bulk water (Dfree). However, as increases, an increasing fraction of the water molecules en-counter network boundaries, thereby restricting diffusion andlowering Dapp to values less than Dfree. At long , all watermolecules experience boundaries, resulting in a limiting valueof Dapp that may be correlated to the root-mean-square (rms)end-to-end distance of the pore space in the hydrogel matrixvia eq 2, in which r is the rms end-to-end distance and td is thevalue of that approaches the limiting value of Dapp.

52

Dapp ) (1 6)td-1r2 (2)

Additional information can be obtained through furtherexamination of the Dapp vs profile. The slope of the initialdecay in Dapp yields information concerning the surface area topore volume ratio (S/Vp), and the magnitude of Dapp at the longtime plateau is indicative of the tortuosity of the medium inwhich diffusion is occurring. A rapid decay of Dapp in the shorttime regime indicates a larger S/Vp, and lower values of Dapp atlong times indicate greater tortuosity.53 For example, PFG-NMRexperiments conducted on idealized systems containing hardspheres provide quantitative values of S/Vp in agreement withthe known values for the beads.54,55 Although quantitativeanalysis of S/Vp for fractal geometries including hydrogelmatrices is currently debated,56 qualitative comparisons havebeen made for hydrogel systems.57

PFG-NMR experiments were first conducted on hydrogelsprepared by crosslinking 0.20 and 0.60 wt % AvP solutionswith 5 mM CaCl2, resulting in the diffusion profiles shown inFigure 9a. rms values of 14 and 13 m were calculated byutilizing the individual Dapp and td values of the 0.20 and 0.60wt % crosslinked systems, respectively. While the calculatedpore sizes of the two systems are similar, the diffusion profilessuggest subtle differences in hydrogel morphology. In com-parison to 0.20 wt % hydrogels, the 0.60 wt % systems exhibita sharper transition as a function of and lower values of Dappat long , indicating greater surface area and tortuosity.

Additional PFG-NMR experiments (Figure 9b) conducted onhydrogels prepared from solutions of moderate ionic strength(0.20 wt % AvP aged in 0.15 M NaCl solutions for 2 and 24 hprior to Ca2+ crosslinking) indicate pores with rms values of 8m. A rapid decay in Dapp as a function of is observed forboth samples, suggesting that high S/Vp ratios are present.

Similar magnitudes of Dapp are obtained at long , suggestingthat tortuosity remains relatively constant in the two systems.Interestingly, the 24 h sample exhibits a second Dapp plateau atlong , suggesting the presence of additional heterogeneitieswithin the hydrogel network.

Large differences in PFG-NMR diffusion profiles betweenhydrogels formed from homogeneous aqueous solutions andsolutions containing 0.15 M NaCl are experimentally observed(Figure 9a,b). Changes in both polymer conformation andsolubility may account for the 40% reduction in rms pore sizenoted for hydrogels formed in the latter case. The PFG-NMRdiffusion profiles also suggest that hydrogels formed in thepresence of NaCl contain greater surface-to-volume ratios(sharper transition in Dapp) and higher levels of tortuosity (lowervalue of Dapp at long ) in comparison to the correspondingaqueous systems.

Hydrogel Morphology. A model of AvP2 hydrogel morphol-ogy consistent with both elastic modulus data and the PFG-NMR studies is depicted in Scheme 2. The model containsmicroscopic aqueous voids surrounded by a polymer rich gelnetwork. Clear aqueous AvP solutions crosslinked by Ca2+ formhomogeneous hydrogel networks with a large amount ofconnectivity between pores. While further studies are neededto elucidate the exact orientation and geometry of the pores,Despang et al.58 have shown that conditions employed duringgelation result in rodlike pores within calcium-alginate hydro-gels. Na+ and K+ ions present in nasal fluid have an additionaleffect on hydrogel morphology. Experimental evidence hasshown that monovalent ions increase AvP association insolution, which at incrementally higher concentration eventuallycauses phase separation. Upon crosslinking, AvP solutionscontaining moderate NaCl concentration (

interchain associations are present in the hydrogel network. Thesharp transition and magnitude of Dapp observed in PFG-NMRstudies suggest that polymer associations increase surface area-to-pore volume ratios and tortuosity within hydrogels.

3.3. Release Profiles of Macromolecular Model Com-pounds. The major objective of this study is to control thediffusion characteristics of crosslinked AvP in order to elicitsustained release of therapeutic agents. In previous sections, wehave shown that controlling AvP concentration, [Ca2+]/[COO-]ratio, and ionic strength prior to or during the crosslinkingprocess results in dramatic changes in physical properties, inparticular viscoelastic behavior and water diffusion. In thissection, we compare the relative rates and extents of release offluorescein labeled 4 kDa (dh ) 3 nm) and 500 kDa (dh ) 27nm) dextran model compounds from Ca2+ crosslinked gelsprepared at low (0.20 wt %) or high (0.60 wt %) concentrationsof AvP2. The Ca2+ concentration for crosslinking was main-tained at 5 mM in each case, a value near that in physiologicalfluids. It should be noted that the labeled model compoundsDex4 and Dex500 (Supporting Information, Figure S6a,b) werechosen as macromolecular model compounds because of theirstability in solution, comparable size to proteins, known effecton pectin gelation,59 and successful use in similar studies.60,61

Figure 10 illustrates the release profiles of the labeled dextransfrom AvP hydrogel matrices as compared to the freely diffusingcontrols, C1 and C2. Curves 1 and 2 demonstrate the rapidrelease of Dex4 and Dex500 respectively in matrices formedfrom 0.20 wt % (dilute) AvP in the presence of Ca2+ only.Cumulative release approaches 100% in 30 h. For curves 3 and4, again from 0.20 wt % AvP hydrogels, but crosslinked in asolution with the Ca2+, Na+, and K+ content of simulated nasalfluid (SNF), release is slower and only reaches 75-80% after96 h, with Dex4 showing only a slightly greater rate and extentof release than the larger Dex500. The effects of increasingAvP2 concentration to 0.60 wt % and size of the dextran onrelease are seen in the final two profiles 5 and 6, againcrosslinked under SNF conditions. Here the larger dextran,Dex500, exhibits a significantly reduced rate and extent ofrelease as compared to Dex4.

In an additional experiment, we determined the amount ofretained dextran that could be released by treating the respectivehydrogel networks with EDTA and then further disrupting theremaining network utilizing mechanical force and mild hy-drolysis (Supporting Information, Figure S7). For example,

5-8% of the dextran is entrapped within crosslinked domainsthat are disrupted by extraction of Ca2+ with EDTA, in both0.20 (Curves 3 and 4) and 0.60 (Curves 5 and 6) wt %hydrogels. However, in curves 5 and 6, an additional 3% ofDex4 and 5% of Dex500 is entrapped within domains, whichremain intact after EDTA exposure.

Theoretical Diffusion Models. To elucidate the diffusionmechanism occurring in AvP hydrogels, the experimental releaseprofiles have been fit to three existing models.62-64 Thesemodels have been previously applied to similar hydrogel systemsincluding alginates and pectins.13,65 Agreement between ourexperimental data and the three diffusion models outlined belowprovides a diagnostic measure of the relative contributions ofFickian diffusion and case II diffusion occurring in Ca2+

crosslinked AvP hydrogels.The first model describes Fickian diffusion based on the

Higuchi equation (eq 3)66

MtM

) kHt12 (3)

where Mt/M represents the fraction of release, t is the releasetime, and kH is the rate coefficient. A fit of experimental datato the Higuchi model indicates diffusion driven release in theabsence of matrix relaxation effects. The characteristic shapeof the experimental Mt/M vs t

1/2 curve is related to thedominant release mechanism, where a sigmoidal departure fromlinearity is indicative of case II diffusion.67

The second model considered was the Ritger-Peppas equa-tion (eq 4), where the exponent n is related to the drug transportmechanism and the shape of the object from which diffusionoccurs.63 In the case of diffusion from a slab, when n ) 0.5,eqs 3 and 4 are equal and Fickian diffusion dominates. Whenn ) 1, eq 4 leads to a description of zero-order release, termedcase II diffusion. Case II diffusion is prevalent when macro-molecular chain relaxations within the hydrogel matrix alter thediffusion rate of the analyte.28 When n is between 0.5 and 1,anomalous or heterogeneous diffusion is suggested.

MtM

) k1tn (4)

The third model, the Peppas-Sahlin equation (eq 5) employsa three-parameter fit, which is utilized to describe anomalousrelease, wherein release profiles are coupled to contributionsfrom both Fickian and case II diffusion. In eq 5, k1 and k2represent the contribution of Fickian diffusion and case IItransport, respectively. In practicality, this model is difficult toanalyze due to the implicit codependence of k1 and k2, howeverreliable solutions for n can be obtained.13

MtM

) k1tn + k2t

2n (5)

To simplify the model, the case where n ) 0.5 has beenexamined from which k1 and k2 can be easily determined.

28

MtM

) k1t12 + k2t (6)

Application of Theoretical Models. Evaluation of experimentaldata relative to the above release models suggests that anincrease in dextran size results in a change in diffusionmechanism. Of the three models examined, the Higuchi equationprovides the best fit to the release profile for curves 1 and 2,suggesting pure Fickian diffusion within hydrogel systemscrosslinked by Ca2+ only. When hydrogels crosslinked under

Figure 10. Cumulative release (%) of 4 kDa (squares) and 500 kDa(circles) dextran as a function of time (h) from 0.20 wt % (black andred symbols) and 0.60 wt % (blue and green symbols) hydrogels. Inthe presence of Na and K only, no gel forms and free diffusion isobserved (curves C1 and C2), while a Ca2+-induced AvP matrixreduces the diffusion rate (curves 1 and 2). When gelled by SNF,diffusion rates are further reduced and dependent on dextran sizeand AvP concentration (curves 3-6).

3284 Biomacromolecules, Vol. 9, No. 11, 2008 McConaughy et al.

SNF conditions are examined (Table 3), it is found that theHiguchi equation still provides the best fit for the release ofDex4 from 0.20 wt % hydrogels (curve 3). With the largerdextran sample curve 4, diffusion is best described by theRitger-Peppas equation. For this system, n ) 0.63, suggestingthat both Fickian and case II diffusion mechanisms are present.Additionally, the Higuchi plot of curve 4 displays sigmoidalcurvature, supporting the conclusion that matrix interactions areinvolved in the diffusion mechanism (Supporting Information,Figure S8). Analysis of 0.60 wt % samples in terms oftheoretical models reveals that the release profiles for both Dex4and Dex500 (curves 5 and 6) are best described by theRitger-Peppas model (Table 3). The values of n suggest thatanomalous diffusion is present in both systems, with thecontribution of matrix relaxations becoming more significantas the size of the dextran species increases.

3.4. Release Mechanisms in View of Hydrogel Charac-teristics. The observed release mechanisms may be explainedby consideration of both the microscopic aqueous pores andthe free pore volume within the segmental structure of the AvP2network. In both the 0.20 and 0.60 wt % systems, aqueous poreswith rms radii between 8 and 13 m have been experimentallyobserved by PFG-NMR. Within these pores, the diffusion of 3and 27 nm dextrans will be unhindered and thus Fickian innature. Fickian diffusion is the dominant component within eachof the AvP hydrogel systems studied, even those that fitanomalous release models, suggesting that a large portion ofdextran diffusion occurs within these micrometer scale pores.

Calculations of the molecular weight between crosslinks (Mc)based on elastic modulus relationships suggest that nanometerscale pores exist within the AvP network, which may hinderthe diffusion of dextran through segmental interactions. Ex-amination of release profiles in terms of theoretical modelsreveals that a significant case II component is involved in thediffusion of Dex4 and Dex500 (Dh, 3 and 27 nm) from 0.60 wt% hydrogels (Table 3), suggesting that segmental (matrix)interactions are present. The elastic modulus-Mc relationshippredicts a distance of 25 nm between crosslinks, consistent withthe increase in case II diffusion observed between 3 and 27 nmdextrans for 0.60 wt % hydrogels (Table 3).

The reduction in dextran release rate observed between thecontrol systems gelled by Ca2+ only and the same 2AvP-Dex4system gelled upon crosslinking with SNF containing Ca2+,Na+, and K+ ions suggests that changes in morphology occurwhen monovalent salts are present. Experimental evidencecollected for 0.60 wt % AvP hydrogels shows that approximately5% of the dextran population is entrapped within crosslinkeddomains that cannot be disrupted by extraction of Ca2+ byEDTA, suggesting that microphase separated domains contribute

to the elastic nature of the hydrogel and affect the release ofdextran. The [Ca2+]/[COO-] ratio in 0.60 wt % systems is lowat physiological concentrations of Ca2+, resulting in a largenumber of free carboxylate functional groups and presumablya significant number of uncrosslinked chain segments. Solid stateNMR experiments conducted by Jarvis et al.68 have shown thatfree pectin chain segments within Ca2+ crosslinked hydrogelsexhibit mobility similar to that in solution.

In view of the experimental evidence, including phasebehavior, elastic moduli and PFG-NMR, it may be concludedthat monovalent electrolyte addition results in chain constrictionand poorer solvation, creating dense AvP2 regions that limitdiffusion by increasing tortuosity. Further support for thisconclusion is drawn from the micrographs shown in Figure 11.AvP hydrogels were stained with ruthenium red in order toobtain a visual diagnostic of polymer homogeneity in thehydrogels. Similarly the fluorescence emission of FITC-dextranwas used to determine the dispersion of dextran throughout thehydrogel matrix. Figure 11a depicts a 0.20 wt % AvP hydrogelcontaining FITC-dextran that was formed on crosslinking withCa2+. The bright field and fluorescence images indicate thatthe dispersion of AvP and dextran are both homogeneousthroughout the hydrogel. In contrast, micrographs taken of AvPhydrogels formed in the presence of 0.15 M NaCl contain

Table 3. Calculated Parameters for Release Models and Goodness of Fit Indicator (AIC) Based on Experimental Release Profiles of 0.20and 0.60 wt % AvP2 Calcium Hydrogels

(3) 2AvP-4Dex-5Ca k1 k2 n AIC

(5) 6AvP-4Dex-5Ca k1 k2 n AIC

Higuchi 2.10 -46.6 Higuchi 1.63 -27.1Ritger-Peppas 2.00 0.51 -41.0 Ritger-Peppas 0.42 0.63 -37.4Peppas-Sahlin 1.83 0.0158 -40.0 Peppas-Sahlin 1.03 0.0157 -25.9

(4) 2AvP-500Dex-5Ca k1 k2 n AIC

(6) 6AvP-500Dex-5Ca k1 k2 n AIC

Higuchi 1.95 -21.4 Higuchi 0.90 -15.1Ritger-Peppas 0.83 0.63 -40.6 Ritger-Peppas 0.33 0.76 -35.3Peppas-Sahlin 1.61 0.0160 -25.0 Peppas-Sahlin 0.87 0.0268 -22.0

Figure 11. Bright field (left image) and fluorescence (right image) of(a) 0.20 wt % AvP2 hydrogels containing FITC-dextran formed bycrosslinking with 5 mM CaCl2, and (b) 0.20 wt % hydrogels formedafter 2 h of exposure to 0.15 M NaCl. The line seen in image (a) isthe sample edge, with the sample lying to the left.

Ca2+ Crosslinked Aloe vera Polysaccharide Hydrogels Biomacromolecules, Vol. 9, No. 11, 2008 3285

polymer rich domains, which contain locally high concentrationsof dextran (Figure 11b).

4.0. Conclusions

The Aloe Vera polysaccharide has been shown to formhydrogels that can be easily tailored for delivery of therapeuticagents when crosslinked by calcium ions. Hydrogel elasticmodulus is independent of AvP molecular weight over the rangeof 200-500 kDa. However, viscoelastic properties are depend-ent upon the concentration of AvP2 and Ca2+ in solution andare also affected by monovalent electrolyte concentrations inAvP2 solutions prior to Ca2+ gelation. Values of G rangingfrom 20-20000 Pa can be obtained by varying the polymerconcentration, the ratio of Ca2+ to COO-, and ionic strength.

As evidenced by changes in the value of the Huggins constantwith monovalent electrolyte addition, segmental association ofAvP occurs in both a concentration and time-dependent manner.Above concentrations of 0.15 M NaCl, phase separation occursin both dilute and concentrated (near C*) AvP solutions. Theobserved increase in modulus values for gels formed in thepresence of monovalent electrolytes is attributed to changes inchain stiffness and solvation as well as local segmentalassociations formed prior to Ca2+ induced gelation. A simplisticmodel (depicted in Scheme 2) has been proposed describingthese matrix changes based on viscoelastic behavior, PFG-NMRstudies of water diffusion, and controlled release of fluoresceinlabeled dextrans of known hydrodynamic volume. The increasedsurface to volume ratio and tortuosity in the segmentally denseregions of the crosslinked matrices appear to be the factorscontributing to the experimentally observed release behavior.

Factors such as polymer stability and hydrogel morphologyare important when considering the design of protein deliverysystems. Experimental evidence suggests that addition ofmonovalent salts to AvP formulations prior to gelation may bebeneficial, increasing elastic modulus and tortuosity whilereducing release rates. However, concentrations must be rela-tively low because high ionic strengths cause phase separationand inhibit Ca2+ induced gelation. Considering these results, itis clear that salt and polymer concentrations must be judiciouslychosen when formulating an in situ gelling therapeutic deliverysystem. It is recommended that an ionic strength less than 0.10M is maintained when AvP concentrations are above 0.10 wt% in order to prevent large scale phase separation and inhibitionof Ca2+ crosslinking. In addition to providing stability and longshelf life, a delivery formulation must also release a precisequantity of protein over a given time interval. Optimal conditionsfor AvP mediated release involve polymer concentrations aboveCe (0.60 wt %), [Ca

2+]/[COO-] ratios that are less than 1, andsolutions at moderate ionic strength.

Acknowledgment. We acknowledge DelSite Biotechnologiesfor financial support, the MRSEC NSF program (DR-0213883),and the NSF Division of Materials Research/Major ResearchInstrumentation award 0079450 for the purchase of the VarianUnity Inova 500 MHz spectrometer. Additionally we thank Dr.Roger Hester for aiding in the theoretical fitting of release data,Dr. Robert Lochhead for providing fruitful discussions concern-ing polysaccharide rheology, and Dr. William Jarrett forassistance acquiring PFG-NMR.

Supporting Information Available. Elastic and viscousmoduli. Example of linear and nonlinear Huggins and Kraemerplots obtained for AvP2. Size distribution of 4 and 500 kDaFITC-Dextrans as determined by dynamic light scattering.

Percent of dextran released during the initial study, after EDTAexposure, and after disruption of phase separated polymerregions. Cumulative release of dextran 4Dex and 500Dex plottedagainst the square root of time for 0.20 and 0.60 wt % AvPhydrogels. This material is available free of charge via theInternet at http://pubs.acs.org.

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