hydrogels in drug delivery - cribme.comcribme.com/cu/data/chemical engineering/tissue... ·...

Hydrogelsindrugdelivery

•Hydrogels:Whattheyareanddifferenttypesof.

•PEGhydrogelsformedbyphotopolymeriza=on.

•Macroscopicproper=esofPEGhydrogels.

•DrugreleasefromPEGhydrogels.

•Macroscopicproper=esofdegradablePEGhydrogels.

•DrugreleasefromdegradablePEGhydrogels.

•Hydrogelsarethree‐dimensionalnetworksofhydrophillic,yetinsolublepolymerchains.

Hydrogels

Silicone‐basedhydrogel

•Whenplacedinanaqueousenvironment,theyswellandareoIencomposedof>90%water(byweight).

•Applica=onsinclude:contactlenses(siliconeorpolyacrylate‐based),diapers(polyacrylate‐based),diet‐aids,treatmentofaneurysms,drug‐deliveryand=ssue‐engineering.

hQp://www.udel.edu/PR/UDaily/2008/jul/gels071807.html

Pep=de‐based

Asmallbiotechcompanyisdevelopingahydrogelthatcanbeplacedonwoundstoimprovehealing.Thisgelreleasesapatenteddrugthatpromotesthewound‐healingprocess.Tobeeffec=ve50%ofthepayloadmustbereleased1haIerapplica=on.Giventhefollowinginforma6onbelowwouldyoupredictthatthedevicewillbesuccessful?Ifnot,whatcouldyoudotoimprovedevicefunc6on?

Ddrug‐water=9.38x10‐5mm2/s

35wt%PEGhydrogel(MW3400)

1mm

2cm

2cm

€

Mesh size, ξ = 41 angstroms

€

Hydrodynamic radius, rs = 40 angstroms

€

∂cA∂t

= Di:p∂ 2cA∂z2

€

cA = c0 t = 0 0 < z < LcA = cS t > 0 z = 0,L∂cA

∂z= 0 t > 0 z = L2

€

cA − csc0 − cs

=4π

12n +1

exp−Di:p (2n +1)2π 2t

L2

n= 0

∞

∑ sin (2n +1)πzL

z=0

z=L

z=L/2

GovEQ.

B.C./I.C.

Solu=on:

AnExpressiontoDescribecA(t,z)asDrugADiffusesthroughoutaHydrogel

Area,A

L

NA

NA

z

c0

cs

z

CA

Time+

€

Mt

M0

=1− 8π 2

1(2n +1)2n= 0

∞

∑ exp −Di:p (2n +1)2π 2t

L2

€

Mt = c0AL − c(z,t)Adz0

L∫

€

For Mt < 0.6M0 : Mt

M0

= 4Di:ptL2π

MassReleased:

TheCummula6veMassofDrugAReleasedfromaPorousPolymerNetwork

z=0

z=L

z=L/2

€

Let M0 = c0ALIni=alDose

Area,A

L

NA

NA

z

Note:AplotofexperimentalMt/M0datavst1/2shouldyieldastraightlinewithaslopethatwouldallowyoutodetermineDi:p(Mt/M0<0.6).

CurrentReleaseprofile

What can be done to improve device function?

Effect of PEG Macromer Weight Percent on Hydrogel Mesh Size

PEGMW3400

K.S. Anseth et al. / Journal of Controlled Release 78 (2002) 199–209 201

Fig. 1. Chemical structures of biodegradable, multifunctional, and photocrosslinkable macromers: (A) PLA-b-PEG-b-PLA macromer and

(B) PLA-g-PVA macromer.

measured using a dynamic mechanical analyzer core with an average molecular weight of 3400 Da,

(Perkin Elmer) with a parallel plate configuration and |2.7 lactic acid repeat units flanking the PEG core,

a ramping stress of 400 mN/min; and their final and 90% acrylation of the end groups. The second

mass was obtained after complete drying in a macromer had a PEG core with an average molecular

vacuum oven. From these measurements, the volume weight of 2000 Da, |2.2 lactic acid repeat unitsswelling ratio, compressive modulus, and mass loss flanking the PEG core, and 70% acrylation of the

were calculated as a function of degradation. end groups. To monitor BSA release as a function of

hydrogel degradation, the gels were placed in vials

2.4. Release studies containing a large excess (10 ml) of pH 7.4 phos-

phate buffered solution at 378C. At specified timeBovine serum albumin (BSA, Sigma) was photo- points, 0.5 ml samples of the vial buffer solution

encapsulated in PLA-b-PEG-b-PLA based hydrogels. were taken and replaced by fresh buffer. After

BSA was added to the macromer solution at con- accounting for dilution caused by previous measure-

centrations up to 4.0 wt%, and photopolymerization ments, protein concentrations were measured with a

of the final mixture produced hydrogel samples |1 Bio-Rad protein assay using the microassay pro-

cm in diameter and 1 mm thick. Release behavior cedure, in which a differential color change of the

from hydrogels formed from two different macrom- dye occurs in response to various protein concen-

ers was examined. The first macromer had a PEG trations. The color change was quantified by measur-

Macromolecules,Vol.34,No.13,2001

Degradable PEG Hydrogels

PEG Lac=cAcid Acrylate

H2O H2O

7048J.Phys.Chem.B,Vol.104,No.30,2000

As discussed during the development of the model, there are

two other fundamental parameters upon which the statistical

model is based. These parameters include (1) the weight percent

of the network contained in the polymer backbone (WPA) relative

to the PLA-b-PEG-b-PLA segments, and (2) the first-order

kinetic rate constant (k!) for the hydrolysis of the PLA units.

The effect of the backbone weight percent on the theoretical

mass-loss predictions is shown in Figure 6. In the physical

system, the backbone weight percent could be increased by

polymerizing a PLA-b-PEG-b-PLA macromer with a lower

molecular weight PEG chain or by copolymerizing a mono-

vinyl species with the divinyl PLA-b-PEG-b-PLA macromer.

All three erosion profiles in Figure 6 undergo reverse gelation

at the same time, since that step depends only upon the number

of cross-links per chain and the kinetic rate constant as shown

in eq 14. The network mass loss is significantly different,

however, before that point. As the backbone weight fraction

decreases, a higher fraction of the network mass resides in the

PLA-b-PEG-b-PLA segments. The PEG chains within these

segments are released at an early time in the network degradation

because they are attached to the gel by only two PLA linkages,

as opposed to the backbone polyacrylate chains that are attached

through 100 PLA-b-PEG-b-PLA cross-links for these particular

systems. Therefore, the curves with lower backbone weight

fractions show higher mass loss percentages at earlier times

because a higher fraction of their weight is located within the

more easily released PLA-b-PEG-b-PLA segments.

The effect of the kinetic parameter, the hydrolysis rate

constant (k!), on mass loss predictions is shown in Figure 7.

This parameter has perhaps the strongest effect on the mass

loss predictions when plotted versus degradation time. This

pseudo first-order rate constant incorporates the true rate

constant along with the water and acid concentrations within

the system. This parameter determines the rate at which the

hydrolysis reaction proceeds and has a direct impact on the rate

of mass loss. The three curves in Figure 7 demonstrate that as

k! increases, the observed rate of mass loss also increases when

other parameters are held constant. The value for the rate

constant also impacts the time at which reverse gelation occurs.

The three curves in Figure 8 share the same characteristic

sigmoidal shape but over drastically different time scales. The

kinetic rate constant (k!) therefore influences only the time scale

of the network erosion while the structural parameters such as

the number of cross-links per kinetic chain (N) and the weight

fraction of the kinetic chains (WPA) determine the characteristic

shape of the hydrogel erosion profile.

Comparison with Experimental Data.The statistical model

that has been developed incorporates the important fundamentals

of the bulk-degradation of a cross-linked hydrogel, and, as

Figures 5 through 7 show, allows one to investigate the

controlling parameters behind the complex process. The measure

of an accurate model, however, is how well it can predict the

degradation behavior of a real system. In Figure 8, mass loss is

again plotted versus degradation time for two PLA-b-PEG-b-

PLA hydrogels polymerized in solution with different concen-

trations of macromer. These degrading hydrogels, therefore,

have the same chemical composition yet different initial cross-

linking densities and microstructures.9 The circles and squares

represent experimental data obtained from the degradation of

these hydrogels in phosphate buffered solution. The solid and

dashed lines represent the mass loss profile predicted by the

statistical model. For each system, the backbone weight fraction

of the network (WPA) was automatically set to match that of

the experimental system, approximately 95% in both cases. The

kinetic rate constants (k!) were calculated using compressive

modulus decay data presented in a previous paper for these same

degrading gels.8 The exponential rate constant of the modulus

Figure 6. Percent mass loss as a function of degradation time forhydrogels with a varying weight percentage in the network backbonechains: (9) 5 wt %; (b) 25 wt %; and ([) 75 wt %. Other modelparameters for all curves: N ) 100 cross-links per backbone chainand k! ) 0.0003 min-1.

Figure 7. Percent mass loss as a function of degradation time forhydrogels with a varying hydrolysis rate constant: (b) k! ) 0.000 07min-1; (9) k! ) 0.0001 min-1; and ([) k! ) 0.0003 min-1. Other modelparameters for all curves: N ) 100 cross-links per backbone chainand WPA ) WPEG ) 50 wt %.

Figure 8. Experimental mass loss data (( 2.0%) as a function ofdegradation time for hydrogels polymerized with varying macromerconcentrations: (9) 25 wt % and (b) 50 wt %. The solid and dashedlines represent the percent mass loss predicted by the statisticalmodel: (dashed) 25 wt % and (solid) 50 wt %. All model parametersused for both fits are given in Table 1.

7048 J. Phys. Chem. B, Vol. 104, No. 30, 2000 Metters et al.

Effect of PEG Macromer Weight Percent on Hydrolysis Rate Constant on Hydrogel Mesh Size

(a)Releasefromthedegradablehydrogelwillbefaster.

WhendrugisreleasedfromadegradablepolymermatrixwilltherateofdrugreleasebefasterorslowerthanthatfromanondegradablematrixpreparedwiththesameMWPEGmacromerandwiththesameweightpercentofPEGmacromerinsolu=on?

(b)Releasefromthedegradablehydrogelwillbeslower.

(c)IfPEGmolecularweightandPEGmacromerweightpercentarethesamethereshouldbenodifferenceintherateofdrugrelease.

Answer:A


with the systematic degradation of network cross-links.This increase in mesh size leads to an increase inmobility for an encapsulated solute as quantified throughits increasing diffusion coefficient (Dg). However, thediffusion coefficient for the trapped solute only increasesto a limiting value, D0.

Solute release from nondegradable hydrogels is knownto be a function of a variety of chemical and physicalparameters that influence the mesh size of the network.As shown by West and Hubbell1 and Lu and Anseth,2the release behavior of PLA-b-PEG-b-PLA hydrogels isalso influenced by their degradation behavior. Equations11 and 12 allow the influence of hydrogel degradationon solute release to be readily quantified and predicted.

The time-dependent function for the solute diffusioncoefficient, eq 12, was used to predict solute release froma degrading PLA-b-PEG-b-PLA hydrogel disk using theone-dimensional diffusional release equation for a uni-formly loaded film.11 Using the calculated degradationbehavior from Figure 3, fractional release profile of BSAfrom the same PLA-b-PEG-b-PLA hydrogel was pre-dicted and is shown in Figure 4. The predicted releaseprofile of an initially similar, yet nondegrading, gel isalso plotted for comparison. This figure illustrates howthe hydrolysis of cross-links within the network in-creases the mesh size and solute diffusion coefficient andenhances the BSA release rate. The overall releaseprofile of the degrading system also has a differentshape than that of an equilibrium-swollen, nondegrad-ing system.

A number of parameters affect the initial structureof a hydrogel, its degradation rate, or both. Most often,these changes are reflected in an increasing or decreas-ing value for k!E or k! () jk!E). For example, increasingthe number of ester bonds per PLA block (j) increasesthe gel degradation rate.4 Also, increasing the extentof functionalization or the macromer concentrationduring network formation leads to lower degradationrates through decreased values of k!E.4 Using eqs 11 and12, Figure 5a shows the effect of increasing the overalldegradation rate constant, k!, on the mesh size of thegel as a function of degradation time. Differences in themesh size and other characteristics of these networksdramatically affect the solute-release profiles predictedfrom these gels, as shown in Figure 5b.Comparison of Experimental and Predicted

Release Profiles. A number of experimental drug-release studies were performed to assess the suitabilityof the scaling laws to correlate drug-release to networkdegradation kinetics. In the first study, two macro-molecular solutes of different molecular weights werereleased from the same degrading network. The volu-metric swelling curves, and therefore degradation ratesof the two systems, are identical. From eqs 3 and 7, thediffusion coefficients of both systems scale identicallywith degradation time, but the absolute values of thediffusion coefficients depend on the solute size (rs). Asrs increases, Dg/D0 decreases, and thus, smaller solutemolecules are released more rapidly. As shown by therelease profiles in Figure 6, this trend is observed forlysozyme (rs ) 16 Å) and BSA (rs ) 35 Å) with thesmaller lysozyme protein being released more quickly.

Figure 3. Mesh size of the degrading hydrogel and thenormalized solute diffusivity as functions of degradation time.

Figure 4. Predicted fractional release of BSA as a functionof time from a nondegrading hydrogel (solid line) and adegrading gel (dashed line) shown in Figure 3. Both gels havean initial mesh size of 41 Å.

Figure 5. (A) Network mesh size and (B) predicted fractionalrelease of BSA as a function of degradation time for gels withvarying degradation rates: (i) k! ) 3.3 ! 10-4 min-1; (ii) k! )3.3 ! 10-5 min-1; (iii) k! ) 3.3 ! 10-6 min-1.

Macromolecules, Vol. 34, No. 13, 2001 PLA-b-PEG-b-PLA Hydrogels 4633


Fig. 1. Chemical structures of biodegradable, multifunctional, and photocrosslinkable macromers: (A) PLA-b-PEG-b-PLA macromer and

(B) PLA-g-PVA macromer.

measured using a dynamic mechanical analyzer core with an average molecular weight of 3400 Da,

(Perkin Elmer) with a parallel plate configuration and |2.7 lactic acid repeat units flanking the PEG core,

a ramping stress of 400 mN/min; and their final and 90% acrylation of the end groups. The second

mass was obtained after complete drying in a macromer had a PEG core with an average molecular

vacuum oven. From these measurements, the volume weight of 2000 Da, |2.2 lactic acid repeat unitsswelling ratio, compressive modulus, and mass loss flanking the PEG core, and 70% acrylation of the

were calculated as a function of degradation. end groups. To monitor BSA release as a function of

hydrogel degradation, the gels were placed in vials

2.4. Release studies containing a large excess (10 ml) of pH 7.4 phos-

phate buffered solution at 378C. At specified timeBovine serum albumin (BSA, Sigma) was photo- points, 0.5 ml samples of the vial buffer solution

encapsulated in PLA-b-PEG-b-PLA based hydrogels. were taken and replaced by fresh buffer. After

BSA was added to the macromer solution at con- accounting for dilution caused by previous measure-

centrations up to 4.0 wt%, and photopolymerization ments, protein concentrations were measured with a

of the final mixture produced hydrogel samples |1 Bio-Rad protein assay using the microassay pro-

cm in diameter and 1 mm thick. Release behavior cedure, in which a differential color change of the

from hydrogels formed from two different macrom- dye occurs in response to various protein concen-

ers was examined. The first macromer had a PEG trations. The color change was quantified by measur-


Fig. 2. Illustration of three different stages during the bulk degradation of a PLA-b-PEG-b-PLA hydrogel network: (A) initial and ideal,

non-degraded PLA-b-PEG-b-PLA network, (B) primary erosion products that are released during degradation, and (C) final degradation

products after complete hydrolysis.

3.2. Hydrogel degradation behavior high degree of swelling, and an almost immeasurable

compressive modulus.

The degradation behavior of several photocros- As indicated by the fitted curves, the swelling of

slinked hydrogels was monitored through mass, the hydrogel increases exponentially with a time

swelling, and mechanical property measurements. constant, t , while the compressive modulus decaysQ

Fig. 3 illustrates the typical in vitro degradation exponentially with a time constant t . These ex-K

behavior for the compressive modulus (K) and perimentally observed exponential changes in the

volume swelling ratio (Q) as a function of degra- macroscopic gel properties with degradation can be

dation time. Immediately following polymerization, explained by the justifiable assumption of a pseudo

the hydrogel displays a high modulus and a low first order kinetic equation for hydrolysis of network

degree of swelling, indicative of a network with a crosslinks [15]. The first order hydrolysis kinetics

relatively high degree of crosslinking. As degra- equation is given by:

dation proceeds, the degradable PLA segments with-dnEin the gel are hydrolyzed homogeneously, cleaving ] 95 2 k n (1)E Edtthe network crosslinks. This process leads to a gel

with a progressively lower crosslinking density, a where n represents the number of moles of degrad-E

Fig. 3. Typical in vitro degradation behavior of a PLA-b-PEG-b-PLA hydrogel: compressive modulus (d) and volumetric swelling ratio

(j). The solid and dashed lines are exponential curves fit to each property with time constants of t 54200 min and t 52000 min.Q K


Degradation of PEG Hydrogels

proportional to Mc, a scaling relationship between thenetwork mesh size and the average molecular weightbetween cross-links is given by:

For high degrees of swelling (!2 < 0.1), the exponentialterm of eq 3 can be neglected to yield the followingsimplified equation for solute diffusivity:

Combining eqs 5 and 6 produces the final scalingrelationship between Mc and the solute diffusion inthese highly swollen gels:

Correlation of Drug Release to DegradationKinetics. The physical network parameters and solutediffusion coefficient were related directly to the kineticsof PLA hydrolysis and degradation time through anunderstanding of degradation behavior of cross-linkedhydrogels and its impact on network microstructure.Experimentally observed exponential changes in themacroscopic properties of PLA-b-PEG-b-PLA hydrogelswith degradation (e.g., swelling and compressivemodulus) can be explained by the justifiable assumptionof a pseudo-first-order kinetic equation for the hydroly-sis of network cross-links.3 The first-order hydrolysiskinetics equation is given by:

where nE represents the number of moles of ester bondsand k!! is the pseudo-first-order reaction rate constantfor the hydrolysis of a single lactide ester linkage.

To reduce the kinetic equation for hydrogel degrada-tion to the simplified form of eq 8, the acid concentrationas well as the water concentration during degradationis assumed constant. All experiments were performedin a phosphate buffered solution, which kept the solu-tion at a constant pH of 7.4 throughout the entiredegradation process. Since the hydrogels used in thisstudy were copolymers with the PLA blocks contributingonly a small fraction to the overall molecular weight,the total acid group concentration in the gels isrelatively low. In addition, the highly swollen nature ofthe gels, due to the presence of the hydrophilic PEGblocks, lowers the concentration of acid species withinthe cross-linked networks even further while allowingfor efficient removal of acidic degradation products.Finally, because of the highly swollen nature of thesegels, the water concentration remains relatively con-stant throughout degradation.

Therefore, as the ester groups and cross-links withinthese hydrogels are hydrolyzed and degradation pro-ceeds, pseudo-first-order kinetics dictate an exponentialincrease in the average molecular weight between cross-links.3 The exponential change in the molecular weightbetween cross-links is related to the physical and kinetic

characteristics of the degrading hydrogel as given by:

where j represents the number of ester bonds per PLAblock in a PLA-b-PEG-b-PLA cross-link and t is thedegradation time.4 The factor of two in the exponentialrate constant of eq 9 occurs because there are two PLAblocks per cross-link (Figure 1). In the PLA-b-PEG-b-PLA network, the degradation of one or more esterbonds leads to the cleavage of a cross-link.

Equations 4 and 9 can be combined to give thefollowing expression for the swelling ratio as a functionof degradation time for the PLA-b-PEG-b-PLA hydro-gels:

Due to the exponential character of the pseudo-first-order hydrolysis kinetics with time, the scaling propor-tionality between Q and Mc given in eq 4 is incorpo-rated directly into the exponent on the right-hand sideof eq 10. The mesh size and solute diffusion coefficientwithin these gels, as functions of degradation time, canalso be scaled:

Equations 10 through 12, therefore, predict an expo-nential dependence for the volumetric swelling ratio,network mesh size, and solute diffusivity in PLA-b-PEG-b-PLA gels as a function of degradation time. Figure 2shows one of many experimental measurements per-formed to verify the exponential behavior of the swellingratio as a function of degradation time in these gels.

In eqs 10 through 12, the PLA block size (j) iscontrolled during macromer synthesis and experimen-tally measured by 1H NMR (Table 1). From the swellingdata, therefore, a value for k!E is readily calculated.Using this value for k!E in eqs 11 and 12, " and (1 -Dg/D0) are predicted readily throughout the course ofhydrogel degradation. As shown in Figure 3, the averagemesh size within the hydrogel increases exponentially

" ) Q1/3(r02)1/2 ! (Mc)

7/10 (5)

Dg

D0) 1 -

rs

" (6)

1 -Dg

D0)

rs

" ! (Mc)-7/10 (7)

dnE

dt) -kE!nE (8)

Figure 2. Experimentally measured volumetric swelling ratiovs degradation time for a typical PLA-b-PEG-b-PLA hydrogel.The dashed line represents the exponential model fit to thedata with a pseudo-first-order hydrolysis rate constant, k!E,of 1.0 " 10-5 min-1.

Mc ! e2jk!Et (9)

Q ! e(6/5)jk!Et (10)

" ! e(7/5)jk!Et (11)

1 -Dg

D0)

rs

" ! e(-7/5)jk!Et (12)

4632 Mason et al. Macromolecules, Vol. 34, No. 13, 2001


Degradation of PEG Hydrogels and Swelling Ratio




A number of parameters affect the initial structureof a hydrogel, its degradation rate, or both. Most often,these changes are reflected in an increasing or decreas-ing value for k!E or k! () jk!E). For example, increasingthe number of ester bonds per PLA block (j) increasesthe gel degradation rate.4 Also, increasing the extentof functionalization or the macromer concentrationduring network formation leads to lower degradationrates through decreased values of k!E.4 Using eqs 11 and12, Figure 5a shows the effect of increasing the overalldegradation rate constant, k!, on the mesh size of thegel as a function of degradation time. Differences in themesh size and other characteristics of these networksdramatically affect the solute-release profiles predictedfrom these gels, as shown in Figure 5b.

Comparison of Experimental and PredictedRelease Profiles. A number of experimental drug-release studies were performed to assess the suitabilityof the scaling laws to correlate drug-release to networkdegradation kinetics. In the first study, two macro-molecular solutes of different molecular weights werereleased from the same degrading network. The volu-metric swelling curves, and therefore degradation ratesof the two systems, are identical. From eqs 3 and 7, thediffusion coefficients of both systems scale identicallywith degradation time, but the absolute values of thediffusion coefficients depend on the solute size (rs). Asrs increases, Dg/D0 decreases, and thus, smaller solutemolecules are released more rapidly. As shown by therelease profiles in Figure 6, this trend is observed forlysozyme (rs ) 16 Å) and BSA (rs ) 35 Å) with thesmaller lysozyme protein being released more quickly.






Degradation of PEG Hydrogels and Mesh size and Diffusion Coefficient

€

1−Dg

D0

=

rsξ

Hydrodynamicradiusofdrug

Meshsizeofthehydrogel

Diffusioncoefficientingel

Diffusioncoefficientinaqueoussolu=on





A number of parameters affect the initial structureof a hydrogel, its degradation rate, or both. Most often,these changes are reflected in an increasing or decreas-ing value for k!E or k! () jk!E). For example, increasingthe number of ester bonds per PLA block (j) increasesthe gel degradation rate.4 Also, increasing the extentof functionalization or the macromer concentrationduring network formation leads to lower degradationrates through decreased values of k!E.4 Using eqs 11 and12, Figure 5a shows the effect of increasing the overalldegradation rate constant, k!, on the mesh size of thegel as a function of degradation time. Differences in themesh size and other characteristics of these networksdramatically affect the solute-release profiles predictedfrom these gels, as shown in Figure 5b.Comparison of Experimental and Predicted

Release Profiles. A number of experimental drug-release studies were performed to assess the suitabilityof the scaling laws to correlate drug-release to networkdegradation kinetics. In the first study, two macro-molecular solutes of different molecular weights werereleased from the same degrading network. The volu-metric swelling curves, and therefore degradation ratesof the two systems, are identical. From eqs 3 and 7, thediffusion coefficients of both systems scale identicallywith degradation time, but the absolute values of thediffusion coefficients depend on the solute size (rs). Asrs increases, Dg/D0 decreases, and thus, smaller solutemolecules are released more rapidly. As shown by therelease profiles in Figure 6, this trend is observed forlysozyme (rs ) 16 Å) and BSA (rs ) 35 Å) with thesmaller lysozyme protein being released more quickly.





Release experiments were also measured from twohydrogels with different extents of acrylate functional-ization, Z. As Z decreases, the initial cross-linkingdensity decreases (increasing the initial swelling ratioand mesh size), as shown in Figure 7a, and the

degradation rate dramatically increases. Both of theseeffects cause an increase in the drug release rate as seenin Figure 7b.

Predictions for the release behavior based on initialcharacteristics of the gels and their degradation ratesare shown by the lines in Figure 7b. In this case,theoretical predictions match experimental release pro-files very well. The agreement between theoretical andexperimental results demonstrates that the scalingequations developed in this work are useful in relatingthe degradation properties of a hydrogel to its soluterelease behavior.

To determine the impact that the initial macromerconcentration has on the release behavior of PLA-b-PEG-b-PLA hydrogels, solute release was measuredfrom a series of hydrogels formed from increasingconcentrations of the same macromer. Increasing thesolvent concentration during polymerization of thesehydrogels has many direct influences on their networkstructure including increased cyclization.12 From thetheoretical kinetic equations for initiation, propagation,and termination, one quickly realizes that the kineticchain lengths will be lowered to some extent with anincrease in solvent concentration. In addition, theinitiation efficiency and the degree of autoaccelerationduring polymerization will also decrease. Although theeffect of each of these changes may be small, lumpedtogether they act to form dramatically different networkstructures with different degradation and solute releasebehaviors.3

The swelling behaviors of gels formed from differentmacromer concentrations are shown in Figure 8a. Asthe macromer concentration in the polymerized solutionis increased from 25 to 50 wt%, the degradation rate,as measured from the exponential swelling curves,doubles even though the initial swelling ratios increaseonly slightly. The experimentally measured soluterelease behavior from these three systems is shown inFigure 8b. These data, along with those presented inFigure 7, demonstrate the effect of hydrogel degradationrate on solute release behavior in the PLA-b-PEG-b-PLAsystems. As the degradation rate increases, the soluteis released at a faster rate, and the shape of the releaseprofile vs degradation time changes. Theoretical predic-tions for this behavior are given by the series of solidlines. The time-dependent solute diffusion coefficient forthese predicted profiles was described and correlatedto the degradation kinetics of each respective systemusing eq 12. Although the qualitative trends of the threedata sets are matched by the model predictions, reason-able fits to the release of all three hydrogels cannot beobtained when the same limiting diffusion coefficient(D0) is used for all systems. Quantitative model fits toall data sets can only be obtained if D0 is allowed todecrease with macromer concentration. These resultsindicate fundamental differences in the network struc-ture that cannot be entirely explained by the currentphysical description of the initial network and itssubsequent degradation.

Conclusions

Scaling laws were developed to understand the solute-release behavior of PLA-b-PEG-b-PLA hydrogels. Thestructural and transport characteristics of these degrad-ing hydrogels, which impact their application as con-trolled release devices, were related to the networkcross-linking density and their hydrolytic degradation

Figure 6. Experimentally measured release of (b) lysozymeand (2) BSA from a hydrogel formed from a 30 wt% solutionof macromer 1 from Table 1. Error bars represent one standarddeviation.

Figure 7. (A) Volumetric swelling ratio and (B) fractionalrelease of BSA as a function of degradation time from two PLA-b-PEG-b-PLA hydrogels with varying extents of acrylatefunctionalization: (b) Z ) 70 ( 5% and (2) Z ) 90 ( 5%. Linesrepresent exponential fits to the swelling data (A) and soluterelease predictions based on scaling equations (B). D0 ) 1.0 !10-5 mm2/s for both curves.

4634 Mason et al. Macromolecules, Vol. 34, No. 13, 2001


Impact of Solute on Kinetics of Drug Release

€

1−Dg

D0

=

rsξ

Hydrodynamicradiusofdrug

Meshsizeofthehydrogel

Diffusioncoefficientingel

Diffusioncoefficientinaqueoussolu=on




A number of parameters affect the initial structureof a hydrogel, its degradation rate, or both. Most often,these changes are reflected in an increasing or decreas-ing value for k!E or k! () jk!E). For example, increasingthe number of ester bonds per PLA block (j) increasesthe gel degradation rate.4 Also, increasing the extentof functionalization or the macromer concentrationduring network formation leads to lower degradationrates through decreased values of k!E.4 Using eqs 11 and12, Figure 5a shows the effect of increasing the overalldegradation rate constant, k!, on the mesh size of thegel as a function of degradation time. Differences in themesh size and other characteristics of these networksdramatically affect the solute-release profiles predictedfrom these gels, as shown in Figure 5b.

Comparison of Experimental and PredictedRelease Profiles. A number of experimental drug-release studies were performed to assess the suitabilityof the scaling laws to correlate drug-release to networkdegradation kinetics. In the first study, two macro-molecular solutes of different molecular weights werereleased from the same degrading network. The volu-metric swelling curves, and therefore degradation ratesof the two systems, are identical. From eqs 3 and 7, thediffusion coefficients of both systems scale identicallywith degradation time, but the absolute values of thediffusion coefficients depend on the solute size (rs). Asrs increases, Dg/D0 decreases, and thus, smaller solutemolecules are released more rapidly. As shown by therelease profiles in Figure 6, this trend is observed forlysozyme (rs ) 16 Å) and BSA (rs ) 35 Å) with thesmaller lysozyme protein being released more quickly.






Impact of Rate Constant for Hydrolysis on Drug Release •Decreasingrateofhydrolysis

Releaseofdrugtetheredtomatrixviaenzyma6callydegradablelinkage

Polyacrylatechains

PEGcross‐links

PEGhydrogel Drug

Releaseisbydiffusion(aIerenzyma=ccleavage)

2. Experimental SectionMaterials. Poly(ethylene glycol) (PEG, Mn ! 10000) was obtained

from Aldrich (St. Louis, MO). Monoacrylate-PEG-N-hydroxysuccin-imide (APEG-NHS, Mn ! 3400) was purchased from Laysan Bio, Inc.(Arab, AL). Fmoc-protected amino acids in their L-configuration aswell as O-benzotriazole-N,N,N!,N!-tetramethyl-uronium-hexafluoro-phosphate (HBTU), 1-hydroxybenzotriazole hydrate (HOBt), and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophos-phate methanaminium (HATU) used for amino acid activation wereobtained from Anaspec (San Jose, CA). MBHA Rink Amide resin waspurchased from Novabiochem (La Jolla, CA). 5(6)-Carboxyrhodamine(ROX) and QXL 610 acid (QXL) were obtained from Anaspec.Fluorescamine was provided by Sigma-Aldrich (St. Louis, MO). Humanneutrophil elastase (HNE) was supplied as a lyophilized powder fromInnovative Research (Novi, MI).

Poly(ethylene glycol) Diacrylate (PEGDA10k) Synthesis. LinearPEGDA10k was synthesized similar to reported literature24 by reactingPEG (Mn ! 10000) with a 8 molar excess of acryloyl chloride in thepresence of triethyleamine (TEA). The reaction was allowed to proceedovernight at room temperature protected from light. The acrylated PEGwas filtered through a bed of alumina to remove the TEA-HClcomplex. Toluene was then removed from the reaction mixture under

rotary evaporation. To obtain pure PEGDA10k, the crude product wasdissolved in methylene chloride and precipitated in cold diethyl ether.The purified product was then filtered and dried in vacuo at roomtemperature. The degree of acrylation was confirmed to be >90% by1H NMR (Supporting Information).

Peptide Synthesis. Peptide sequences (Table 1) were synthesized(Applied Biosystem 433A Peptide Synthesizer) using solid phase Fmocchemistry on a MHBA Rink Amide Resin (!0.7 mmol/g resinsubstitution). Peptides were cleaved from their solid support usingtrifluoroacetic acid (TFA)/triisopropylsilane (TIS)/water (95/2.5/2.5 v/v)and allowed to react at room temperature for 2 h. The reaction wasfiltered and the filtrate precipitated and washed (3") in chilled diethylether. Peptides were purified by semipreparative reversed phase HPLC(Waters Delta Prep 4000) using a 70 min linear (5-95%) gradient ofacetonitrile in 0.1% trifluoroacetic acid. Peptide purity was confirmedby analytical reversed phase HPLC C18 column and matrix-assistedlaser desorption ionization time-of-flight mass spectrometry (AppliedBiosystem DE Voyager).

Kinetic Analysis of Substrate Degradation. Degradation kineticparameters were determined using a fluorescamine fluorometric assay.25

To measure accurately the concentrations of cleaved peptide fragments,the N-terminal amines of the HNE substrate peptides were capped with

Scheme 1. Photopolymerization of PEGDA with an Acrylated HNE-Sensitive Substrate

Table 1. HNE-Sensitive Peptides Synthesized Showing Point Variations in the P1 and P1! Positionsa

a Arrow indicates cleavage location. Unless otherwise noted, the default amino acid for the P1 position was Val and Gly for the P1! position. Non-native amino acid abbreviations represent Abu ) aminobutyric acid, Nva ) norvaline, and Nle ) norleucine.

Human Neutrophil Elastase Biomacromolecules, Vol. 10, No. 6, 2009 1485

Acrylate

PEG

Enzyme‐sensi=vepep=de

Drug

Whichofthefollowingchangeswillincreasetherateofdrugreleasefromahydrogel?

a.Alteringdegradablepep=desequencetodecreasekcatfortheenzyme‐substratereac=on

b.IncreasingtheMWofthePEGmacromer

c.Increasingenzymeconcentra=oninthegel

d.Alloftheabove

e.Twooftheabove

Answer:E

Youcanalterkcatinthiscasebyincorpora=nganarginineresidueintothesequence.

acetic anhydride. Peptides were dissolved in reaction buffer (50 mMHEPES + 150 mM NaCl pH 7.4) at varying concentrations and HNEwas added (30nM). The reaction mixture was sampled at 5-min intervalsand allowed to further react with fluorescamine (2 mg/mL in acetoni-trile) for determining the concentrations of cleaved peptides (fluores-cence was detected at !excitation ) 380 nm, !emission ) 460 nm, Perkin-Elmer Wallac Victor2 1420 Multilabel Counter). Cleavage productconcentration was determined using presynthesized peptide fragmentsas an external calibration. Michaelis-Menten enzyme kinetic analysiswas performed and specificity constants (kcat/Km) were determined usingnonlinear regression analysis (Graphpad Prism 5). Substrate cleavagesite was confirmed by RP-HPLC analysis. The peptide fragments wereisolated and analyzed with electrospray ionization mass spectrometryto determine the amino acid composition within the fragment.

Synthesis of HNE-Cleavable FRET Substrate. The peptideK(ROX)AAPVVRGGGK(QXL) was synthesized as follows (wherearrow indicates cleavage location). Fmoc-Lys(Mtt)-OH was allowedto couple to the resin (HATU/N,N!-diisopropylethylamine (DIPEA))for 2 h. The resin was then treated with 1.8% TFA in dichloromethanefor 30 s, repeated nine times26 to selectively remove the Mtt protectinggroup on Lys. A ninhydrin test was performed to confirm the completeremoval of the Mtt protecting group. QXL was then reacted to thedeprotected !-amino group (HATU/DIPEA) on Lys for 2 h. The resinwas then thoroughly washed with DMF and placed on the ABI 433APeptide Synthesizer for automated couplings. After peptide synthesis,Fmoc-K(Mtt)AAPVRGGGK(QXL)-resin was removed from the instru-ment and the Mtt group selectively deprotected as described above.ROX was reacted to the N-terminal Lys !-amino group using HATU/DIPEA coupling chemistry. The terminal Fmoc group was manuallyremoved (20% piperidine in DMF). Finally, the product was cleavedfrom the resin and purified as described above. The product wasconfirmed using MALDI-MS.

Visualization of HNE Activity in PEG Hydrogel. Acrylate-PEG-NHS (5 equiv) was reacted to the N-terminal amine of the FRETsubstrate in 0.1 M sodium phosphate buffer pH 8.0 for 4 h protec-ted from light. The desired product (Acryl-PEG-K(ROX)-AAPVVRGGGK(QXL)) was isolated using RP-HPLC and resulted ina lyophilized powder. Upon HNE dictated cleavage, the QXL quencheris able to diffuse away, leaving fluorescent ROX that provides spatialevidence of HNE activity within our gels. FRET hydrogels were formedfrom a precursor solution of 10 wt % PEGDA10k in phosphate buffer(PBS), 0.025 wt % of the photoinitiator 2-hydroxy-1-[4-(hydroxyethox-y)phenyl]-2-methyl-1-propanone (I-2959, Ciba-Geigy), and 4 mM acryl-PEG-K(ROX)AAPVRGGGK(QXL), exposed to 365 nm ultraviolet

light for 10 min. Gels were treated with 1 µM HNE and 3-dimensionalfluorescence image stacks, spanning the thickness of the hydrogel, werecaptured at predetermined time points using confocal microscopy (ZeissPascal LSM 5). ROX was excited using a 543 nm helium-neon laserand fluorescence was collected using a 560 nm long pass filter.

HNE Dictated Release From PEG Hydrogel.Two HNE substratesanalyzed using the solution phase assay were further studied in animmobilized hydrogel system. Peptides AAPVVRGMG and AAP(Nva)VGGMG were acrylated with conjugation to APEG-NHS and purifiedas described previously. Methionine residues were substituted forcysteine residues, as used previously, to prevent thiol-acrylate reactionduring photopolymerization. Hydrogels were formed via free radicalphotopolymerization from the macromer solution containing 10 wt %PEGDA10k, 5 mM APEG-peptide, and 0.025 wt % I-2959 understandard conditions described previously. Cylindrical disks (diameter) 5 mm, thickness ) 600 µm) were formed using a biopsy punch.Roughly 85% of the APEG-peptide was incorporated during thephotopolymerization, which is comparable to previous literature usingsimilar reaction conditions.27 This was determined by swelling the gelsin buffer for 24 h and exposing the supernatant to HNE (1 µM). Thereaction was allowed to proceed for 2 h to ensure complete substratecleavage. Fluorescamine was added and the amount of peptide in thebuffer solution was determined using an external calibration withthe peptide fragment. Gels (n ) 3) were then transferred to 100 µL offresh HEPES buffer and 1 µM HNE was added. At predetermined timepoints, samples were analyzed for peptide fragment release usingfluorescamine. Buffer and enzyme was replenished at each time point.

3. Results and Discussion

3.1. Solution Phase Enzyme Kinetic Analysis of HNESubstrates. To tailor drug delivery in enzyme responsivematerials, it is important to characterize the kinetic rate at whichthe enzyme is breaking down its respective substrate. HNEsubstrates were synthesized with point variations in the P1 andP1! amino acid positions with the goal of manipulating thereaction kinetics. Rational design was used to vary residueswithin these locations. Previous literature reports that HNEspecificity for its substrate depends greatly on S!-P! interac-tions.28 Therefore, we examined a charged amino acid (Arg), aneutral, hydrophilic residue (Gln), a hydrophobic, aliphaticamino acid (Leu), and a hydrophobic, aromatic residue (Phe)in the P1! location (Table 1). Figure 1a shows that hydrophobic

Figure 1. Solution phase hydrolysis of peptides by HNE. The values of kcat and Km for point variations in the P1! (a) and P1 (b) substratepositions. Kinetic constants for the control substrate Ac-YAAPVVGGCG were Km ) 160 ( 20 µM and kcat ) 2.68 ( 0.08 s-1. Arrow indicatescleavage location. (c) Representative Michaelis-Menten plot. (d) Representative HPLC chromatogram showing pure peptide (dashed line) andpeptide and HNE reaction mixture (solid lines).

1486 Biomacromolecules, Vol. 10, No. 6, 2009 Aimetti et al.

Biomacromolecules,Vol.10,No.6,2009

2. Experimental SectionMaterials. Poly(ethylene glycol) (PEG, Mn ! 10000) was obtained

from Aldrich (St. Louis, MO). Monoacrylate-PEG-N-hydroxysuccin-imide (APEG-NHS, Mn ! 3400) was purchased from Laysan Bio, Inc.(Arab, AL). Fmoc-protected amino acids in their L-configuration aswell as O-benzotriazole-N,N,N!,N!-tetramethyl-uronium-hexafluoro-phosphate (HBTU), 1-hydroxybenzotriazole hydrate (HOBt), and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophos-phate methanaminium (HATU) used for amino acid activation wereobtained from Anaspec (San Jose, CA). MBHA Rink Amide resin waspurchased from Novabiochem (La Jolla, CA). 5(6)-Carboxyrhodamine(ROX) and QXL 610 acid (QXL) were obtained from Anaspec.Fluorescamine was provided by Sigma-Aldrich (St. Louis, MO). Humanneutrophil elastase (HNE) was supplied as a lyophilized powder fromInnovative Research (Novi, MI).

Poly(ethylene glycol) Diacrylate (PEGDA10k) Synthesis. LinearPEGDA10k was synthesized similar to reported literature24 by reactingPEG (Mn ! 10000) with a 8 molar excess of acryloyl chloride in thepresence of triethyleamine (TEA). The reaction was allowed to proceedovernight at room temperature protected from light. The acrylated PEGwas filtered through a bed of alumina to remove the TEA-HClcomplex. Toluene was then removed from the reaction mixture under

rotary evaporation. To obtain pure PEGDA10k, the crude product wasdissolved in methylene chloride and precipitated in cold diethyl ether.The purified product was then filtered and dried in vacuo at roomtemperature. The degree of acrylation was confirmed to be >90% by1H NMR (Supporting Information).

Peptide Synthesis. Peptide sequences (Table 1) were synthesized(Applied Biosystem 433A Peptide Synthesizer) using solid phase Fmocchemistry on a MHBA Rink Amide Resin (!0.7 mmol/g resinsubstitution). Peptides were cleaved from their solid support usingtrifluoroacetic acid (TFA)/triisopropylsilane (TIS)/water (95/2.5/2.5 v/v)and allowed to react at room temperature for 2 h. The reaction wasfiltered and the filtrate precipitated and washed (3") in chilled diethylether. Peptides were purified by semipreparative reversed phase HPLC(Waters Delta Prep 4000) using a 70 min linear (5-95%) gradient ofacetonitrile in 0.1% trifluoroacetic acid. Peptide purity was confirmedby analytical reversed phase HPLC C18 column and matrix-assistedlaser desorption ionization time-of-flight mass spectrometry (AppliedBiosystem DE Voyager).

Kinetic Analysis of Substrate Degradation. Degradation kineticparameters were determined using a fluorescamine fluorometric assay.25

To measure accurately the concentrations of cleaved peptide fragments,the N-terminal amines of the HNE substrate peptides were capped with

Scheme 1. Photopolymerization of PEGDA with an Acrylated HNE-Sensitive Substrate

Table 1. HNE-Sensitive Peptides Synthesized Showing Point Variations in the P1 and P1! Positionsa

a Arrow indicates cleavage location. Unless otherwise noted, the default amino acid for the P1 position was Val and Gly for the P1! position. Non-native amino acid abbreviations represent Abu ) aminobutyric acid, Nva ) norvaline, and Nle ) norleucine.

Human Neutrophil Elastase Biomacromolecules, Vol. 10, No. 6, 2009 1485

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