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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.
[email protected]. 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.
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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
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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.
Ca2+ Crosslinked Aloe vera Polysaccharide Hydrogels
Biomacromolecules, Vol. 9, No. 11, 2008 3281
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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.
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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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BM8008457
Ca2+ Crosslinked Aloe vera Polysaccharide Hydrogels
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