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The effect of the linker on the hydrolysis rate ofdrug-linked ester bondsRonald G.Schoenmakers a,*,Petra van de Wetering a ,Donald L.Elbert b,1,Jeffrey A.Hubbell aaInstitute for Biomedical Engineering,ETH and University of Zurich,Moussonstrasse 18,CH-8044Zurich,SwitzerlandbDepartment of Biomedical Engineering,Washington University,Campus Box 1097,One Brookings Drive,St.Louis,MO 63130-4899,USAReceived 13August 2003;accepted 9December 2003AbstractTailoring the length of a sulfide containing linker adjusts the hydrolysis of a drug-linked ester bond to values appropriate for once-a-week administrations.A model drug of paclitaxel was coupled using a hydrolyzable linker to a poly(ethylene glycol)macromonomer,via a conjugate addition reaction between a thiol and an acrylamide.The macromonomers were synthesized in three steps with an average overall yield of 70%.By changing the length of the linker from 3-sulfanylpropionyl to 4-sulfanylbutyryl,the half-life time of the release of the drug could be increased from 4.2F 0.1to 14.0F 0.2days.Drug-containing hydrogels were prepared by radical photopolymerization of these macromonomers with either the 3-sulfanylpropionyl or the 4-sulfanylbutyryl linker.The release of the drug from these hydrogels followed similar trends as the release of the drug from the soluble polymer–drug conjugates.The synthetic methodology employed does not involve the use of coupling reagents in the final conjugation between the drug and the polymer,excluding the presence of potential toxic residuals.The conjugation method is relatively simple and is applicable to nearly any hydroxyl-containing drugs.D 2004Elsevier B.V .All rights reserved.Keywords:Prodrugs;Drug release;Conjugate addition;Hydrogels;Poly(ethylene glycol)1.IntroductionWater-soluble polymers can be conjugated to drugs to achieve a number of beneficial modifica-tions of the drug’s pharmacokinetics and pharmaco-distribution.With regard to macromolecular drugs,the clearance of protein drugs by the reticulo-endo-thelial system can be substantially reduced by con-jugation to water-soluble polymers; e.g.a 10-fold increase in the plasma circulation time of intrave-nously administered interferon-a 2b was observed for poly(ethylene glycol)(PEG)conjugated interferon-a 2b relative to its native form [1].For low molecular weight drugs,the fraction of drugs directed toward the hyper-permeable vasculature found in tumors relative to the less permeable vasculature found in most normal tissues can be increased by conjugation to water-soluble polymers; e.g.conjugation of the0168-3659/$-see front matter D 2004Elsevier B.V .All rights reserved.doi:10.1016/j.jconrel.2003.12.009*Corresponding author.Tel.:+41-1-6344556;fax:+41-1-632-1214.E-mail address:ronald.schoenmakers@mat.ethz.ch (R.G.Schoenmakers).1Fax:+/locate/jconrelJournal of Controlled Release 95(2004)291–300potent anticancer drug doxorubicin to N-(2-hydroxy-propyl)methacrylamide was observed to increase the fraction of drug targeting tumors,decrease systemic toxicity,and increase therapeutic efficacy[2].With hydrophobic low molecular weight drugs,conjuga-tion to water-soluble polymers can increase solubility and thus enable administration without the use of hydrophobic carrier vehicles like liposomes[3], emulsions[4],and poly(lactic-co-glycolic acid)-based nanoparticles[5];e.g.increased solubility and circu-lation times have been observed in water-soluble polymer-conjugates of doxorubicin[6],camptothecin [7],daunorubicin[8],podophyllotoxin[9].However, an important prerequisite of the resulting polymer–drug conjugates(polymeric prodrugs)is that the drug is regenerated in an acceptable time frame,and no toxic moieties are released or formed due to this in vivo transformation.It has been shown that an effectively permanent conjugation of PEG to pacli-taxel leads to higher circulation times,however,the PEG-conjugate is less active than the native com-pound[10].In addition to applications in polymeric prodrugs and polymer-linked drug conjugates with enhanced solubility,water-soluble polymers can also be used to prepare cross-linked drug delivery systems.The poly-meric carrier can be cross-linked to obtain a three-dimensional depot,from which the drug release pro-file is dependent on the hydrolysis rate of the linker between the drug and the polymer,thermodynamic partitioning of the drug within the cross-linked poly-mer,and the kinetics of diffusion of the drug from the pared to incorporation of drugs within a depot by physicochemical methods,covalent conju-gation provides an additional means by which to control the release rate,potentially decoupling the rate of release from the physicochemical character-istics of the drug and the depot matrix.In both soluble and cross-linked drug-conjugated polymers,of course the nature of the drug–polymer link plays a key role in the rate of drug release by generation of the native drug.Here,we focus on non-enzymatic hydrolytic release, e.g.not considering approaches developed for colonic release using aro-matic azo bonds that can be cleaved by microbial azoreductase activities[11]and intracellular release via lysosomally activated proteases[2,12].We further focus on hydrolytic mechanisms that are not triggered by biological changes in pH,as have been achieved using hydrazone linkages[13,14]and with selected orthoesters[15].Due to their relative sensitivity to hydrolysis,esters have generated substantial interest. However,the ester linkages that have been reported either hydrolyze too slowly for many extracellular applications at physiological pH[16]or too quickly [17–19].Here we present a novel ester linker,formed between a hydroxyl-containing drug and a mercap-toacid.We show that the number of methylene units that intervene between the ester link and the sulfide in the final product modulates the hydrolytic sensi-tivity of the ester in a rather sensitive manner,two yielding hydrolysis over a few days,and three yielding hydrolysis over a few weeks.The hydroly-sis of the ester that has a sulfide at the h or g position yields the native hydroxylated drug.The synthetic route utilized permits facile incorporation into cross-linked gel formulations,and we present an example using PEG-based cross-linked elastic gels. As a therapeutically interesting example,we select a model compound of the low aqueous-solubility anticancer drug paclitaxel,3-(S)-benzoylamino-2-(R)-hydroxy-3-phenyl-propionic acid methyl ester, employing its hydroxyl group in the side chain as the drug conjugation site.2.Materials and methodsAll reagents were obtained from commercial sour-ces and used as purchased.Dichloromethane(DCM) and N,N-dimethylformamide(DMF)were dried on molecular sieves(4A˚).Reactions were monitored by thin layer chromatography(TLC)on Fluka precoated silica gel60F-254(0.25mm)plates.Spots were visualized with UV light,ninhydrin or Cl2-TDM (N,N,N V,N V-tetramethyl-4,4V-diaminodiphenylmethane). Column chromatography was performed on Kieselgel 60(Machery-Nagel,CH).1H NMR spectra were recorded on a Bruker(300.1MHz)spectrometer and chemical shifts are given in ppm(d)relative to TMS as internal standard.13C NMR spectra were recorded on the same spectrometer operating at75.5MHz. Mass spectrometry was carried out on an IonSpec HiResMALDI using2,5-dihydroxybenzoic acid as matrix.R.G.Schoenmakers et al./Journal of Controlled Release95(2004)291–300 2922.1.Syntheses2.1.1.Preparation of3-(S)-benzoylamino-2-(R)-hy-droxy-3-phenyl-propionic acid methyl ester(1) 3-(S)-Benzoylamino-2-(R)-hydroxy-3-phenyl-pro-pionic acid methyl ester(1)was prepared from3-(S)-benzoylamino-2-(R)-hydroxy-3-phenyl-propionic acid in methanol using thionyl chloride according to the method described by Brenner and Huber [20].1H-NMR(CDCl3)d3.38(s,1H,HCO H),3.83(s, 3H,OC H3),4.63(d,1H,C H OH),5.72and5.83(d d, 1H,C H Ph),7.01(d,1H,N H),7.25–7.53(m,23H, Ar),7.76(d t,2H,Ar).13C-NMR(CDCl3)d53.2(C HPh),54.8(H C OH), 73.2(O C H3),126.9–138.7(Ar),166.9(C OOMe), 173.4(C ONH).2.1.2.Preparation of3-(S)-benzoylamino-3-phenyl-2-(R)-(3-sulfanyl-propionyloxy)-propionic acid methyl ester(4a)2.1.2.1.3-Tritylsulfanylpropionic acid(2a).A solu-tion of0.87ml(10mmol)3-mercaptopropionic acid and2.86g(11mmol)triphenylmethanol in40ml DMF was stirred for30min at60j C.After the solution was cooled to ambient temperature,1.43ml (11.4mmol)boron trifluoride ethyl etherate was added and the reaction mixture was stirred at60j C for4h.The solution was concentrated in vacuo and the residue was dissolved in500ml5%NaHCO3.The aqueous solution was washed with250ml diethyl ether,acidified to pH3using1M KHSO4,and extracted twice with500ml ethyl acetate.The com-bined ethyl acetate fractions were washed with250ml brine and dried over Na2SO4.The product was obtained as a white solid in90%yield.1H-NMR(CDCl3)d2.23(t,2H,C H2S),2.46(t, 2H,C H2COOH),7.23–7.43(m,15H,Trt).2.1.2.2.3-(S)-benzoylamino-3-phenyl-2-(R)-(3-trityl-sulfanyl-propionyloxy)-propionic acid methyl ester (3a).A solution of120mg(0.4mmol)of1and 280mg(0.8mmol)3-tritylsulfanyl-propionic acid (2a)in4ml DCM and1ml DMF was cooled to0 j C.Then126A l(0.8mmol)diisopropylcarbodiimide (DIPC)and a few crystals of dimethylaminopyridine (DMAP)were added and the reaction was stirred at0j C for2h.After the reaction was complete based on TLC analysis of the reaction mixture,the solvents were removed in vacuo,and the crude product was dissolved in50ml DCM.The organic solution was washed with1M KHSO4,5%NaHCO3,and brine, consecutively,and dried over Na2SO4.After purifica-tion by column chromatography(silica,eluent:ethyl acetate/hexane1:2v/v),a white solid was obtained with a yield of74%.1H-NMR(CDCl3)d2.00–2.55(m,4H,C H2S andC H2COO),3.67and3.73(s,3H,OC H3),5.51and5.38(d,1H,C H O),5.72and5.83(d d,1H,C H Ph),6.35and6.96(d,1H,N H),7.15–7.59(m,23H,Ar and Trt),7.96and7.76(d t,2H,Ar).13C-NMR(CDCl3)d27.7and26.7(C H2S),35.7 and33.5(C H2COO),52.8and52.9(C HPh),53.3and 53.4(H C O),67.0and67.1(C(Ph)3Trt),74.8and74.5 (O C H3),126.6–144.7(Ar and Trt),165.4and166.9 (C OOMe),168.2(C OOCH),170.5(C ONH).2.1.2.3.3-(S)-benzoylamino-3-phenyl-2-(R)-(3-sul-fanyl-propionyloxy)-propionic acid methyl ester (4a).A solution of126mg of the trityl protected compound3a(0.2mmol)in DCM was added to10ml of a trifluoroacetic acid(TFA),triisopropylsilane (TIS),and water(95:2.5:2.5v/v/v)solution,and the reaction mixture was stirred at room temperature for1 h.The solvents were removed in vacuo,and the product was obtained after column chromatography (silica,eluent:ethyl acetate/hexane1:2v/v)in a quantitative yield.1H-NMR(CDCl3)d1.59–1.64(m,1H,S H),2.67–2.77(m,4H,C H2SH and C H2COO),3.76(s,3H, OC H3),5.50(d,1H,C H O),5.90(d d,1H,C H Ph), 7.04(d,1H,N H),7.26–7.80(m,8H,Ar),7.81(d t, 2H,Ar).13C-NMR(CDCl3)d19.6(C H2SH),38.2 (C H2COO),52.9(C HPh),53.4(C HOCO),74.6 (O C H3),126.6–137.4(Ar),166.9(C OOMe),168.3 (C OOCH),170.4(C ONH).MALDI-TOF MS:m/z=410.1(M+Na).2.1.3.Preparation of3-(S)-benzoylamino-3-phenyl-2-(R)-(4-sulfanyl-butyryloxy)-propionic acid methyl es-ter(4b)2.1.3.1.4-Tritylsulfanylbutyric acid(2b).A solution of0.48g(2mmol)4,4-dithiodibutyric acid in freshlyR.G.Schoenmakers et al./Journal of Controlled Release95(2004)291–300293distilled dioxane(20ml)was stirred under a nitrogen atmosphere.Then0.52ml(2.1mmol)tributylphos-phine and50A l water were added and the reaction mixture was stirred for4h at room temperature.After removing the solvents in vacuo the crude product was dissolved in25ml DMF together with1.15g(4.4 mmol)triphenylmethanol,and the solution was stirred for30min at60j C.After the solution was cooled to ambient temperature,580A l(4.6mmol)boron tri-fluoride ethyl etherate was added and the reaction mixture was stirred overnight at80j C.The solution was concentrated in vacuo and the product purified by column chromatography(silica,eluent:ethyl acetate/ hexane1:1v/v).After recrystallization from ethyl acetate/hexane the product was obtained as white needles with a yield of50%.1H-NMR(CDCl3)d1.67(m,2H,CH2C H2CH2), 2.27(m,4H,C H2S and C H2COOH),7.15–7.48(m, 15H,Trt),10.49(br s,1H,COO H).13C-NMR(CDCl3)d23.8(CH2C H2CH2),31.1 (C H2S),32.9(C H2COOH),66.7(C(Ph)3Trt), 126.6–144.8(Ar Trt),179.0(C OOH).2.1.3.2.3-(S)-benzoylamino-3-phenyl-2-(R)-(4-trityl-sulfanyl-butyryloxy)-propionic acid methyl ester (3b).This reaction of133mg(0.44mmol)of1 and177mg(0.49mmol)4-tritylsulfanylbutyric acid (2b)with76A l(0.49mmol)DIPC and a few crystals of DMAP was performed as described for compound 3a.The product was obtained as a white solid in77% yield after column chromatography(silica,eluent: ethyl acetate/hexane1:3to1:2v/v).1H-NMR(CDCl3)d1.60(m,2H,CH2C H2CH2), 2.20(m,2H,C H2COO),2.33(m,2H,C H2S),3.74(s, 3H,OC H3),5.40(d,1H,C H O),5.84(d d,1H,C H Ph),6.96(br d,1H,N H),7.16–7.54(m,23H,Ar and Trt),7.79(d t,2H,Ar).13C-NMR(CDCl3)d23.7(CH2C H2CH2),31.1 (C H2S),32.7(C H2COO),52.8(C HPh),53.4 (H C OCO),66.7(C(Ph)3Trt),74.3(O C H3),126.5–144.8(Ar and Trt),166.8(C OOMe),168.4(C OOCH), 171.7(C ONH).2.1.3.3.3-(S)-benzoylamino-3-phenyl-2-(R)-(4-sul-fanyl-butyryloxy)-propionic acid methyl ester (4b).The removal of the trityl group from the thiol was performed as described for compound4a.After column chromatography(silica,eluent:ethyl acetate/hexane1:3to1:2v/v)the product was obtained as a white solid in an81%yield.1H-NMR(CDCl3)d1.29(t,1H,S H),1.89(m,2H, CH2C H2CH2),2.53(m,4H,C H2SH and C H2COO),3.77(s,3H,OC H3),5.47(d,1H,C H O),5.89(d d,1H,C H Ph),6.99(d,1H,N H),7.28–7.57(m,8H,Ar),7.80 (m,2H,Ar).13C-NMR(CDCl3)d23.6(CH2C H2CH2),28.7 (C H2SH),32.1(C H2COO),52.9(C HPh),53.4 (C HOCO),74.4(O C H3),126.5–137.5(Ar),166.9 (C OOMe),168.4(C OOCH),171.7(C ONH).MALDI-TOF MS:m/z=424.1(M+Na).2.1.4.Conjugate addition and cross-linking reactions 2.1.4.1.Conjugate addition reaction between4a and PEG-3400diacrylamide.To a solution of10mg(25 A mol)4a and361mg(100A mol)PEG-3400diacry-lamide(5)in1ml dry methanol was added3.3A l(25 A mol)triethanolamine.PEG-3400diacrylamide was synthesized from PEG-3400diamine according to the procedure described by Elbert and Hubbell[21].The reaction mixture was stirred in the dark under an argon atmosphere at room temperature for4h.The solvent was removed by a nitrogen gas flow,and the residue was dissolved in1ml50mM aqueous acetic acid and purified using a PD10column(Pharmacia). The fractions containing PEG were collected and lyophilized.The product(6a)was obtained in a yield of91%(based on PEG-3400diacrylamide).2.1.4.2.Conjugate addition reaction between4b and PEG-3400diacrylamide.The conjugation of11mg (25A mol)4b with361mg(100A mol)PEG-3400 diacrylamide(5)was performed analogous to the conjugation reaction described as above resulting in macromonomer6b(90%yield).2.1.4.3.Formation of cross-linked PEG biomaterial containing covalently bound3-(S)-benzoylamino-2-(R)-hydroxy-3-phenyl-propionic acid methyl ester.To100A l of a10mg mixture of PEG-3400 diacrylamide(5)and PEG-3400a-monoacrylamide, N-mono(3-(S)-benzoylamino-2-(R)-hydroxy-3-phe-nyl-propionic acid methyl ester)(6)from above in a well of a96well plate was added N-vinyl pyrrolidone (0.35A l)and Eosin Y(1A l of a10mM solution in HEPES buffered saline,pH7.4).This solution wasR.G.Schoenmakers et al./Journal of Controlled Release95(2004)291–300 294exposed to visible light at about500nm(75mW/cm2) for1min,producing a cross-linked network.2.2.Release2.2.1.Release from PEG-linked conjugates in solutionA solution of50mg PEG-linked conjugate in1ml 10mM phosphate buffered saline(PBS),pH7.4was incubated at37j C.Periodically,samples of50A l were withdrawn to which1A l1M HCl was added to adjust the pH to4.The samples were stored atÀ30j C until analyzed.At the end of the release study100%release was determined by adjusting the pH to10.Because the methyl ester of the model drug was hydrolyzed during the release experiment,the formation of both 3-(S)-benzoylamino-2-(R)-hydroxy-3-phenyl-pro-pionic acid methyl ester and its acid were determined in order to obtain the total release of model drug from the polymer.The amount of the compounds was measured with HPLC using a reversed phase C18 column(Nova-Pak R C18, 3.9Â150mm,Waters Associates),a Waters2690Separation Module (Waters Associates)and a Waters996Photodiode Array Detector(Waters Associates).A linear gradient of0.1%TFA in water/acetonitrile75:25v/v to25:75 v/v in17.5min at a flow rate of1ml/min was used as elution system.The chromatograms were analyzed with Waters Millennium R32software(version 3.05.01).2.2.2.Release of conjugate from a photopolymerized hydrogelA10%w/v photopolymerized hydrogel from10 mg PEG-linked conjugate was incubated at37j C in1 ml PBS,pH7.4.Periodically,samples of50A l were withdrawn and replaced by fresh buffer.The amount of released compound was measured with HPLC as described above.2.3.Molecular modelingStructures were built using Chem3D7.0(Cam-bridgeSoft).The starting structures were optimized with a series of restricted Hartree-Fock calculations. The geometries of the structures were first optimized with the semi-empirical PM3method[22,23]using MOPAC[24].The obtained structures were then optimized using ab initio methods subsequently employing the small3-21G basis set[25]and the larger6-31G*basis set[26]using GAMESS[27].The atomic charges of the final conformations were cal-culated with the6-31G*basis set.2.4.CMC measurementsThe critical micelle concentrations(CMC)of the macromonomer solutions were determined using a pyrene fluorescence assay.Fluorescence measure-ments were performed on a Perkin Elmer LS50B fluorimeter.A stock solution of0.1M pyrene was made in THF.By diluting the stock solution to2Á10À6 M pyrene in PBS buffer(pH7.4),the pyrene working solution was obtained.Polymer solutions with varying concentrations were made in PBS.Just before the measurement,equal volumes of pyrene working so-lution and polymer solution were combined in a cuvette and stirred.For the measurement of pyrene emission spectra,pyrene was excited at339nm and emissions at373and383nm were taken as first and third vibronic peaks.3.Results and discussionThe conjugation of PEG,as an example of a water-soluble polymer that is synthetically convenient due to its well-defined,telechelic nature,to a model hydroxyl-containing drug,namely3-(S)-benzoyla-mino-2-(R)-hydroxy-3-phenyl-propionic acid methyl ester,is outlined in Fig.1.The first step in the conjugation was coupling of the linker,a protected mercaptoacid,to the hydroxyl moiety of the model drug.We employed the commercially available mer-captoacid3-mercaptopropionic acid and4-mercapto-butyric acid,which was obtained after reduction of its disulfide,4,4V-dithiodibutyric acid.Before coupling the linker to the model drug,the thiol group of the linker was protected with a trityl group using triphe-nylmethanol and boron trifluoride ethyl etherate.The protected linker was subsequently coupled to the hydroxyl group of the model drug via an esterifica-tion using DIPC in the presence of DMAP.Also,the trityl-protected mercaptoacetic acid was used,how-ever,the coupling product with the model drug could not be obtained.After removing the trityl group usingR.G.Schoenmakers et al./Journal of Controlled Release95(2004)291–300295TFA,TIS and water (95:2.5:2.5v/v/v)and purifica-tion,compound 4was attached to PEG-diacrylamide via a conjugate addition reaction between the thiol and acrylamide using triethanolamine as a base.The polymer–drug conjugate was obtained in 3steps with an overall yield of about 70%.One favorable feature of our synthetic scheme is that the drug is coupled to the linker first,allowing the linker-conjugated precursor to be purified by means available to small molecules.In the final step,the linker-conjugated drug is further conjugated to the polymer via a conjugated addition reaction,also known as Michael-type addition.This reaction be-tween polymer and derivatized drug does not require coupling reagents and generates no leaving group,residuals of either of which are often difficult to remove from the macromolecular product.Thus,in contrast to the methods in which the drug is subjected to several modification reactions before obtaining a polymer–drug conjugate,the presence of other reac-tants than used in the Michael-type addition can be excluded in our system.A further convenient feature of the synthetic route we chose may be seen in the generation of the cross-linked solid final form.By using an excess of PEG-diacrylamide in the conjugate addition to the linker-conjugated drug,a mixture of a heterobifunctional macromonomer 6with a homobi-functional PEG-diacrylamide (5)is obtained.Thus,the drug-linked macromonomer 6may be copolymer-ized with the PEG-diacrylamide that is also produced to obtain a final cross-linked PEG-based hydrogel.The aqueous solubilities of these polymer–drug conjugates 6a and 6b were similar to that of the PEG-diacrylamide employed.The release of the model drug in vitro under physiological conditions was first inves-tigated from the soluble,i.e.non-cross-linked,drug-conjugated polymer to allow measurement of release without concerns of the effect of the gel environment upon hydrolysis.The measured release profiles are shown in Fig.2and can be well described by first-order kinetics.Fig.2clearly shows the influence of the linker on the release rate,which is reflected in the exponential estimated half-lives for hydrolysis of 4.2F 0.1and 14.0F 0.2days for the 3-sulfanylpro-pionyl and 4-sulfanylbutyryl linkers,respectively.In order to get a better insight into the relation between structure and hydrolysis kinetics,molecular modeling experiments were performed.Besides the 3-sulfanylpropionyl and 4-sulfanylbutyryl linker,also the 2-sulfanylacetyl linker,which is expected to have a shorter half-life than the other two linkers,was included in the modeling experiments.To investigate the probability of a hydroxyl attack on the carbonyl group of the ester of interest,the atomic charge on the carbon atom of this carbonyl group was calculated.As can be seen in Fig.3,the atomic charge on the carbon atom decreases with an increasing number of methy-lene units between the ester bond and the sulfide.From these results it can be expected that the hydro-lysis rate of the ester decreases with an increasing linker length.This is in accordance with the observed difference in hydrolysis half-lives between the 3-sulfanylpropionyl and 4-sulfanylbutyryl linker.As described above,use of a slight excess of PEG-diacrylamide in the synthesis scheme results in a mixture of the macromonomer of the drug andPEG-Fig.1.Synthesis of the model drug-conjugated macromonomer,which can then be further polymerized to form a high polymer or cross-linked hydrogel (m =2:3-sulfanylpropionyl linker,m =3:4-sulfanylbutyryl linker).R.G.Schoenmakers et al./Journal of Controlled Release 95(2004)291–300296diacrylamide,which can then be polymerized into a cross-linked hydrogel drug delivery matrix by radical polymerization or a conjugate addition reaction with a compound containing multiple thiol groups.In this study,hydrogels were made from both3-(S)-ben-zoylamino-2-(R)-hydroxy-3-phenyl-propionic acid methyl ester-PEG conjugates6a and6b,by radical photopolymerization using the method described by Elbert and Hubbell[21].After preparation at a total solids content of10%w/v in water,the obtained hydrogels swelled an additional22F2%during the incubation in buffer at37j C to an equilibrium solids content of8%w/v.The release of the model drug conjugated into the hydrogels via both the3-sulfa-nylpropionyl or4-sulfanylbutyryl linker is presented in Fig.4.As observed for the PEG conjugates in solution,the release of the model drug is dependent on the linker length.However,in the case of the gels, the release profiles for both linkers could only be described by first-order kinetics until about45%of the total release.Above45%,the release gradually starts to deviate from a first order profile.This Fig.3.Optimized structures of the different linkers(A:2-sulfanylacetyl,B:3-sulfanylpropionyl,and C:4-sulfanylbutyryl)attached to the model drug3-(S)-benzoylamino-2-(R)-hydroxy-3-phenyl-propionic acid methyl ester,and the atomic charge on the carbon atom of the esterbond. Fig.2.Release of model drug from the PEG conjugates in solution(PBS,pH7.4,37j C,n=3,F S.D.n:3-sulfanylpropionyl linker;b:4-sulfanylbutyryl linker.R.G.Schoenmakers et al./Journal of Controlled Release95(2004)291–300297suggests a physicochemical effect that modulates release,in addition to hydrolysis from the linker,such as diffusion through the gel phase after hydro-lysis.Because both gels (with the 3-sulfanylpropionyl and with the 4-sulfanylbutyryl linker)have practically identical architecture,the release curves of the model drug from the gels approach and overlap each other in the course of time.It has been reported by several groups that PEG modified with hydrophobic end groups forms micelles [28–30].The drug-conjugated PEG macromonomer might also be able to form micelles consisting of a hydrophobic core in which the drug is concentrated,and a surrounding hydrophilic PEG layer.Due to the hydrophobic environment of the core the hydrolysis rate could be lower compared to the release rate in a hydrophilic environment like in a hydrogel.Whether the soluble polymer–drug conjugates were able to form micelles was checked with a pyrene assay.Indeed,the presence of hydrophobic nanodomains was observed,both in the case of the drug-conjugated macromonomer and the PEG-diacrylamide,with CMC values in the range 0.03–0.06mM,similar to values reported previously for PEGs monofunctional-ized with hydrophobic groups [28,29].However,comparing the release curves of the model drug fromthe polymer–drug conjugates in solution (Fig.2)and the release of the model drug from the hydrogels (Fig.4),it can be seen that release from the hydrogels is not significantly faster than release from the soluble polymer–drug conjugates for this effect to be consid-ered an important modulator of hydrolysis rate.This can be explained by the exchange of the polymers between the micelle and sol phases,as described by Yamamoto et al.[31].Alternatively,the macromono-mer might be polymerized in the micellar phase.Both cases result in a similar exposure of the ester bond in the hydrogel and in the soluble polymer–drug conju-gate to the medium in the time frame of the release.4.ConclusionsEster linkages between drugs and polymeric carries have been utilized previously in polymeric prodrugs,however at physiological,extracellular pH,particular esters with desirable hydrolysis rates have not been described to enable formulations suitable for once-a-week pletely aliphatic esters of paclitaxel are hydrophobic and extremely stable [16].On the other hand,when the linker between drug and PEG is e.g.an amino acid (derivative)orsuccinicFig.4.Release of model drug from PEG-diacrylamide-cross-linked hydrogels formed by photopolymerization (PBS,pH 7.4,37j C,n =3,F S.D.n :3-sulfanylpropionyl linker;b:4-sulfanylbutyryl linker.R.G.Schoenmakers et al./Journal of Controlled Release 95(2004)291–300298acid,the half-lives are in the order of several hours to a few days,too short for many applications[17–19].Here,we present a conjugation methodology using a drug-linked ester bond with hydrolysis half-life times in the order of a few days to a few weeks. 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