New poly(amidoamine)s containing disulfide linkages in their main chain

New Poly(amidoamine)s Containing Disulfide Linkages inTheir Main Chain

ELISA EMILITRI, ELISABETTA RANUCCI, PAOLO FERRUTI

Dipartimento di Chimica Organica e Industriale, Via Venezian 21, 20123 Milan, Italy

Received 9 September 2004; accepted 15 October 2004DOI: 10.1002/pola.20599Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Novel poly(amidoamine)s (PAAs) containing disulfide linkages regularlyarranged along their backbones were synthesized by the stepwise polyaddition of2-methylpiperazine to N,N�-bis(acryloyl)cystamine (BACy1) or N,N�-bis(acryloyl)-(L)-cystine (BACy2). Both bisacrylamides had, in turn, been obtained by the reaction ofacryloyl chloride with the corresponding amines. All the products were characterizedwith 1H and 13C NMR spectroscopy, and the average molecular weights of the polymerswere determined by size exclusion chromatography. Both PAAs showed different sol-ubility properties. In particular, PAA-Cy1, derived from BACy1, was sparingly solublein water, whereas PAA-Cy2, derived from BACy2, was very soluble in aqueous media.The polymerization rates were investigated with 1H NMR spectroscopy. In both cases,the experimental data were consistent with pseudo-second-order kinetics. The calcu-lated kinetic constants were 5.96 � 10�3 and 5.90 � 10�2 min�1 L mol�1 for thepolyaddition of BACy1 and BACy2, respectively. The observed hydrolytic degradationrate of PAA-Cy2 in a pH 7.4 tris(hydroxymethyl)aminomethane (TRIS) buffer wascomparable to that of conventional amphoteric PAAs, that is, PAAs containing carboxylgroups in their repeating unit. Degradation experiments carried out in the presence of2-mercaptoethanol with both PAAs demonstrated that the disulfide groups contained inits repeating units were susceptible to reductive cleavage in the presence of thiols.© 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1404–1416, 2005Keywords: degradation; functionalization of polymers; hydrophilic polymers; kinet-ics (polym.); poly(amidoamine)s; soluble drug carriers

INTRODUCTION

A major requirement for a polymer to be used asa constituent of a drug-release system is the abil-ity to give a predictable response to changes inthe biological environment,1 such as conforma-tional adjustments to pH variations or the degra-dation of polymer–drug linkages at predeter-mined pH levels, or the ability to interact withspecific cell recognition sites.

Polymeric release systems containing disulfidelinkages in their structure, with the disulfide re-duction triggering the release of the active sub-stance, represent valuable alternative routes toconventional hydrolytically degradable systems,in that the drug can be released from the polymerin physiologically reducing environments. In oraladministration, disulfide linkages can deliver theactive substance to the colon, in which reductionis caused by bacterial flora, whereas they are notaffected by the pH change in the digestive tract.2,3

Colon delivery results in an increased therapeuticeffect because of local accumulation of the drug inthe case of treatment of local gastrointestinaltract diseases, such as ulcerative colitis and

Correspondence to: P. Ferruti (E-mail: [email protected])Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 43, 1404–1416 (2005)© 2005 Wiley Periodicals, Inc.

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colonal diseases. Side effects caused by the sys-temic circulation of drug are also reduce becauseno absorption of the drug occurs before it reachesthe colon. For parenteral administration, drugrelease is triggered via disulfide reduction by en-dogenous thiols,4 which include glutathione(GSH), present in a concentration of approxi-mately 10 �M in human plasma,5 free reducedhomocysteine (0.1–0.35 �M),6 and free cysteine(ca. 5 �M).7 Polymeric drug-delivery systems con-taining disulfide linkages are considered particu-larly useful for the delivery of peptide and peptidomimetic therapeutic agents, which suffer frommajor limitations because of degradation andpoor absorption in the gastrointestinal tract.Some authors have proposed poly(ethylene glycol)(PEG) conjugates with bioactive molecules, suchas peptides or enzymes,8,9 drugs,10 or lipo-somes,11,12 in which the PEG–substrate linkage isa disulfide group, which has been proved stable ata neutral pH but is unstable in physiologicallyrelevant reducing environments. Moreover, thedisulfide linkage might be especially useful fordrug delivery into cells because of the strongerreducing environment within cells than in extra-cellular fluids. The presence of a reducible disul-fide bond is, for instance, considered a prerequi-site for the cytotoxicity of immunotoxins, a classof therapeutic agents composed of monoclonal an-tibodies linked to a proteic toxin and directed totumor-cell-associated antigens.13,14

Poly(amidoamine)s (PAA)s are a family of wa-ter-soluble, biomimetic, and biodegradable poly-mers obtained by the polyaddition of primary orsecondary aliphatic amines to bisacrylamides (seeScheme 1).15,16

Many PAAs have properties that make themparticularly suitable for use as soluble drug car-riers. Targeting moieties are easily added as sidesubstituents.17 The hydrophilicity of most of themallows the solubilization of hydrophobic drugs,whereas pendant functions introduced to theirrepeating units allow the attachment of drug moi-eties, targeting groups, or diagnostic moieties.PAAs generally show low cytotoxicity or hemo-lytic activity at pH 7.4 and conformational andactivity changes as an effect of pH. AmphotericPAAs, that is, PAAs containing carboxylic func-tions as side chains, also show stealth properties,that is, the ability to avoid uptake by the cells ofthe reticuloendothelial system.18 As a result, theyexhibit passive targeting to tumors by an en-hanced permeation and retention effect (EPR) ef-fect.15 PAA-grafted proteins, such as albumin,

have also been prepared, with advantage beingtaken of the presence of free amino groups in theprotein.19

On the basis of this premise, we thought itinteresting to report in this article on the synthe-sis and characterization of novel PAAs containingdisulfide linkages regularly arranged along theirpolymeric backbones. These PAAs, PAA-Cy1 andPAA-Cy2, were obtained by the stepwise polyad-dition of 2-methylpiperazine (2MP) to N,N�-bis(ac-ryloyl)cystamine (BACy1) and N,N�-bis(acryloyl)-(L)-cystine (BACy2), respectively (see Scheme 2).

The use of BACy1 has already been reported inthe literature for the synthesis of polyacrylamidegels whose shape can be altered after polymeriza-tion (shape imprinting); it is incorporated as areversible crosslinker with smaller amounts of apermanent crosslinker.20 BACy1 has also beenused21 in the synthesis of polyacrylamide gelscarrying disulfide groups that upon reduction pro-vide sulfhydryl groups useful for the immobiliza-tion of biologically active substances. Both BACy1and BACy2 have also been used as comonomers inthe synthesis of iron-binding polymers intendedfor oral administration to reduce the gastrointes-

Scheme 1. Synthesis of PAAs.

NEW POLY(AMIDOAMINE)S 1405

tinal absorption of dietary iron in related meta-bolic disorders.22

PAA-Cy1, a cationic PAA scarcely soluble inaqueous media at neutral pH or higher, and PAA-Cy2, an amphoteric PAA completely soluble inwater at all pH values, represent the first exam-ples of a new family of PAAs containing disulfidelinkages in their main chain and capable of open-ing the way to the selective modification of pep-tides or proteins through cysteine oxidation. Theresults of exchange experiments carried out in thepresence of 2-mercaptoethanol as a reducingagent demonstrate that the disulfide functioncontained in each repeating unit of PAA-Cy1 andPAA-Cy2 might exhibit specific degradation path-ways in biologically relevant reducing environ-ments. Therefore, these PAAs have potential forthe preparation of polymeric drug-release formu-lations for colon delivery. Hydrolytic degradationexperiments, carried out in the presence of water-soluble PAA-Cy2 at pH 7.4 and 37 °C, indicatethat this PAA exhibits a hydrolytic degradationbehavior comparable to that of conventional am-photeric PAAs.

EXPERIMENTAL

Materials

Cystamine dihydrochloride (97.0%), L-cystine(�99.0%), NaOH (99%), LiOH � H2O (99%), andacryloyl chloride (97%) were purchased fromFluka and used without further purification. 2MPwas purchased from Fluka and recrystallizedfrom n-heptane before use. D2O (99.9%), CD3OD

(�99.8%), and CDCl3 (�99.8%), stabilized over asilver coil, were purchased from Aldrich Co.

Instruments and Methods

The 1H and 13C spectra were acquired on aBruker Avance 400 spectrometer operated at400.133 (1H) and 100.00 MHz (13C).

Size exclusion chromatography (SEC) chro-matographs of PAA-Cy2 were obtained with TSK-gel G4000 PW and TSK-gel G3000 PW columnsproduced by TosoHaas. The two columns wereconnected in series, and the mobile phase was atris(hydroxymethyl)aminomethane (TRIS) buffer(pH 8 or 10, flow rate � 1 mL/min; HPLC pump515, Waters); the UV detector was a Watersmodel 486 operated at 230 nm, and the refractivedetector was a Waters model 2410. The sampleswere prepared in the TRIS buffer with a 1% con-centration in the polymer. The molecular weightdeterminations were based on a calibration curveobtained with pullulan standards. SEC traces ofPAA-Cy1 were obtained with Phenomenex Phe-nogel 500-, 103-, and 104-Å columns, connected inseries, with a Knauer UV detector operated at 254nm and at a flow rate of 1 mL/min. The mobilephase was a 9:1 chloroform/methanol mixture,and the polymer concentration was about 1 wt %.The molecular weight determinations were basedon a calibration curve obtained with polystyrenestandards.

The intrinsic viscosities were obtained in wateror methanol, at 30 °C, with an Ubbelohde capil-lary viscometer in dilute solutions (0.1–0.6 g/dL).

Scheme 2. Structure of PAA-Cy1 and PAA-Cy2.

1406 EMILITRI, RANUCCI, AND FERRUTI

Synthesis of BACy1

Cystamine dihydrochloride (11.6 g, 0.05 mol) wasadded to a four-necked, 500-mL flask equippedwith a stirrer, a thermometer, and two 25-mLdripping funnels and was dissolved in water (50mL). After the mixture cooled to 0–5 °C, an acry-loyl chloride dichloromethane solution (9.3 g, 0.1mol; 10 mL) and an aqueous NaOH solution (8 g,0.2 mol; 20 mL) were added simultaneously anddropwise under stirring over a total time span of1 h; the temperature was kept at 0–5 °C. Afterthe addition was complete, the reaction mixturewas stirred at room temperature for 2 h more.The raw product was recovered according to thefollowing procedure. The organic phase was sep-arated from the aqueous one, and the latter wasextracted with dichloromethane (3 � 100 mL).The collected organic phases were dried overNa2SO4. The suspension was finally filtered off,and the solid was discarded. The solvent was re-moved at 40 °C in vacuo. The raw bisacrylamidewas purified by crystallization from a 1:2 ethylacetate/heptane mixture.

Yield: 9.12 g (70%). mp: 120–123 °C. ELEM.ANAL. Calcd. for C15H28N4O2S2: C, 49.97%; H,7.87%; N, 15.54%; S, 17.79%. Found: C, 49.76%;H, 7.89%; N, 15.45%; S, 17.63%.

Synthesis of BACy2

In a four-necked, 500-mL flask equipped with astirrer, a thermometer, and two 25-mL droppingfunnels, cystine dihydrochloride (12.1 g, 0.05 mol)and methoxy hydroquinone (100 mg) were dis-solved in water (50 mL). After the mixture cooledto 0–5 °C, a 10 M acryloyl chloride solution indichloromethane (10 mL) and a 1 NaOH aqueoussolution (40 mL) were added simultaneously anddropwise under stirring over a total time span of1 h; the temperature was kept at 0–5 °C. Afterthe addition was complete, the reaction mixturewas stirred at room temperature for an additional2 h.

The raw product was recovered according tothe following procedure. The aqueous phase wasseparated from the organic one, and its pH wasadjusted to 1–2 with a 2 M HCl aqueous solution.A translucent suspension was thus obtained. Wa-ter was removed by freeze drying, and a whitesolid was obtained was suspended in acetone. Theresidual insoluble NaCl was filtered off and dis-carded. Acetone was then removed by vacuumvaporization, and the solid product that was ob-

tained was purified by crystallization from ace-tone/diethyl ether.

Yield: 9.6 g (55%). mp: 115–121 °C. ELEM. ANAL.Calcd. for C17H28N4O2S2 � H2O: C, 43.76%; H,6.48%; N, 12.01%; S, 13.74%. Found: C, 43.72%;H, 6.59%; N, 11.99%; S, 13.69%.

Polymerization of BACy1 with 2MP (PAA-Cy1)

BACy1 (0.520 g, 2.0 mmol) and 2MP (0.206 g, 2.0mmol) were dissolved in a 6:1 (v/v) methanol/water mixture (0.6 mL) under an N2 atmosphere.The reactant solution was maintained at roomtemperature for 24 h. After the reaction was com-plete, the pH of the polymerization solution wasadjusted to 1–2 with an aqueous 2 M HCl solu-tion. The solution was then precipitated in a 10-fold excess of acetone. The solid product that wasobtained, corresponding to the hydrochloride saltof PAA-Cy1, was dried in vacuo to a constantweight.

Yield: 0.68 g (85%). 1H NMR (CD3OD, �): 1.30,1.44 (3H totally, CH3), 2.82–2.82 (4H, broad m,CH2CONH), 2.88 (4H, t, CH2SS), 3.54 (4H, t,CH2NHCO), 3.50 (4H, CH2N), 3.10–3.80 (broadm, piperazine ring CH2), 3.80–3.90 (1H, pipera-zine CHOCH3), 3.60–3.90 (1H, piperazineCHOCH3). 13C NMR (CDCl3, �): 22.0 (piperazineCH3), 29.5 (CH2CONH), 37.12 (CH2SS), 38.3(CH2CCONH), 40.8 (CH2NHCO), 47.5 (m, ringCH2), 52.0 (m, ring CH), 170.32 (CONH). Intrin-sic viscosity of the free base in methanol at 25 °C:0.20 dL/g. Solubility of the free base: soluble inmethanol, diethylene glycol, chloroform, andDMSO; insoluble in water, acetone, and ether.Number-average molecular weight (Mn): 15,000.Weight-average molecular weight (Mw): 26,000.

Polymerization of BACy2 with 2MP (PAA-Cy2)

BACy2 (1.00 g, 2.9 mmol), 2MP (297 mg, 2.9mmol), and LiOH � H2O (122 mg, 2.9 mmol) weredissolved in water (0.5 mL) under an N2 atmo-sphere. The mixture was allowed to react for 24 h.The polymeric product was recovered and purifiedas described for PAA-Cy1.

Yield: 1.25 (85%). 1H NMR (CDCl3, �): 1.0 (3H,CH3), 2.5, 2.8 (2H each, CH2CCONH), 2.2, 3.2(1H, piperazine CHOCH3), 2.4, 3.2 (1H each, ax-ial and equatorial ring CH), 2.7, 3.1 (1H each,axial and equatorial ring CH), 2.9, 3.2 (d, 2H,CH2SS), 3.10, 3.40 (1H each, axial and equatorialring CH2OCH), 3.40 (2H, CH2CO), 4.47 (1H,CHCOOH). 13C NMR (CDCl3, �): 18.0 (piperazine


CH3), 30.5 (CH2CCONH), 40.1 (CH2SS), 42.9,48.2, 52.7 (ring CH2), 51.0 (CH2CONH), 54.1(CHOCOOH), 56.0 (ring CH), 174 (CONH), 177(COOH).

Both spectra refer to the hydrochloride salt ofPAA-Cy2. All assignments were obtained by H–Hcorrelation spectroscopy and J1 C-H correlationspectroscopy.

Intrinsic viscosity in a pH 7.40 phosphatebuffer at 25 °C: 0.23 dL/g. Solubility of the freebase: soluble in aqueous media at different pHvalues; insoluble in organic solvents. Mn: 10,000.Mw: 21,000.

Kinetic Study of the Polymerization Reaction ofBACy1 and 2MP (PAA-Cy1)

BACy1 (235 mg, 1.15 mmol) and 2MP (115 mg,1.15 mmol) were dissolved under nitrogen in a 6:1CD3OD/D2O mixture to produce a 1.0 M concen-tration of both monomers. The reaction mixturewas then rapidly transferred to an NMR tube,and the tube was sealed. 1H NMR spectra wererecorded at regular times. The progress of thepolymerization reaction was followed through themonitoring of the reduction in the area of thepeaks at 5.8 and 6.2 ppm, with respect to thedouble-bond hydrogens. The conversion was ex-perimentally determined from the ratio of thepeak integral of the selected diagnostic peaks tothat of the resonance peak placed at 1.0 ppm,with respect to the CH3 group, taken as an inter-nal standard.

Kinetic Study of the Polymerization Reaction ofBACy2 and 2MP (PAA-Cy2)

BACy2 (391 mg, 1.19 mmol), LiOH � H2O (50 mg,1.19 mmol), and 2MP (119 mg, 1.19 mmol) weredissolved under nitrogen in D2O to produce a 1.0M concentration of both monomers. The mixturewas then transferred to an NMR tube, and thetube was sealed. The progress of the polymeriza-tion reaction was followed through the monitor-ing of the reduction of the area of the diagnosticpeaks placed between 6.0 and 6.4 ppm, with re-spect to the double-bond hydrogens. The conver-sion was experimentally determined from the ra-tio of the peak integral of the selected diagnosticpeaks to that of the resonance peak placed at 4.3ppm, with respect to the CHOCOOH group,taken as an internal standard.

Hydrolytic Degradation Studies of PAA-Cy2

PAA-Cy2 (200 mg) was dissolved under a nitrogenatmosphere in a pH 7.4 TRIS buffer (20 mL), andthe obtained homogeneous solution was main-tained at 37 °C under a nitrogen atmosphere withstirring. Aliquots of the solution (50 �L) weretaken at regular times and analyzed at by SECoperated in the aqueous phase (as discussed pre-viously).

Reductive Degradation Studies of PAA-Cy2

PAA-Cy2 (200 mg, 0.43 mmol of the repeatingunit) was dissolved under a nitrogen atmospherein a pH 7.4 TRIS buffer (20 mL), and 2-mercap-toethanol (160 mg, 2.05 mmol) added. The ob-tained homogeneous solution was maintained at37 °C under a nitrogen atmosphere with stirring.Aliquots of the solution (50 �L) were taken atregular times and analyzed by SEC operated inthe aqueous phase (as discussed previously).

Reductive Degradation Studies of PAA-Cy1

PAA-Cy1 (200 mg, 0.55 mmol of the repeatingunit) was dissolved in an HPLC-grade 9:1 dichlo-romethane/methanol mixture (20 mL). The homo-geneous solution that was obtained was main-tained at 37 °C under a nitrogen atmosphere withstirring, and then 2-mercaptoethanol (215 mg,2.75 mmol) WAS added. Aliquots of solutions (50�L) were taken at regular intervals and analyzedby SEC operated in the organic phase (as dis-cussed previously).

RESULTS AND DISCUSSION

Synthesis of the Monomers

The BACy1 and BACy2 monomers were synthe-sized according to a classic reaction pathway in-volving N-acylation of the amine groups of cysta-mine and L-cystine, respectively, with acryloylchloride (see Scheme 3) under conventionalSchotten–Baumann conditions. A few differenceswere adopted for the overall experimental proce-dures. For instance, for BACy1, a 2:1 acryloylchloride/cystamine ratio was used, whereas a2.5:1 acryloyl chloride/L-cystine ratio was re-quired for BACy2. Moreover, the synthesis ofBACy2 was carried out in the presence of a radi-cal inhibitor, 4-methoxy-hydroquinone, to preventundesired vinyl polymerization of the monomer.


Crude BACy1, which was not soluble in aqueousmedia, was recovered after the complete reactionin the methylene chloride phase, whereas crudeBACy2, obtained initially as a sodium salt, wasrecovered in the aqueous phase and then con-verted into its free carboxylic acid with HCl.BACy2 was recovered by filtration, dried, andeventually separated from residual NaCl by ex-traction with acetone.

Both BACy1 and BACy2 were characterizedwith 1H and 13C NMR. The resulting spectra arereported in Figures 1 and 2 for BACy1 and Fig-ures 3 and 4 for BACy2, together with assign-ments. The spectroscopic data were in full agree-ment with the proposed structures.

Synthesis of the Polymers

The aim of this article is to report the synthesis oftwo novel homopolymeric PAA structures con-taining disulfide linkages in their repeating unitswith potential as bioactive and biocompatiblepolymers. These PAAs, PAA-Cy1 and PAA-Cy2,were obtained by the polyaddition of 2MP to twodifferent bisacrylamides containing disulfidelinkages in their structure: a neutral bisacrylam-ide, BACy1 (see Scheme 4), and a bisacrylamidecontaining two carboxyl functions in its structure,BACy2 (see Scheme 5).

The rationale for the design of PAA-Cy2, awater-soluble amphoteric PAA capable of under-going reductive cleavage in biological environ-

ments, is based on our previous knowledge on theunique behavior of amphoteric PAAs as bioactiveand biocompatible polymers. Previous studies inthis field have shown that the cytotoxicity ofPAAs is strictly correlated to their polycationicnature.21 More specifically, amphoteric PAAs aremuch less cytotoxic than poly(L-lysine) and poly-(ethylenimine), and their toxicity can be corre-lated with the basicity constant of the secondamino group present in the repeating unit. Theleast cytotoxic amphoteric PAAs exhibit, in fact,an isoelectric point lower than the physiologicalpH. Depending on their acid–base properties,that is, on their isoelectric point, PAAs that areprevailingly cationic at pH 7.4 show higher cyto-toxicity. In contrast, those that are prevailinglyanionic at pH 7.4 are not cytotoxic. In particular,a PAA, ISA23, obtained by the polyaddition of2MP to 2,2-bisacrylamidoacetic acid has provedpromising for biomedical applications because ofits low toxicity, stealth properties, and transfec-tion ability.21 The design of PAA-Cy1, which issparingly soluble in water and nonamphoteric,represents an interesting model hydrophobicPAA, whose repeating unit can be used to addbiologically cleavable disulfide linkages to differ-ent hydrophilic PAA structures and tune the hy-drophilic–hydrophobic balance.

The polymerization reaction leading to PAAs isgenerally carried out in solvents carrying mobilehydrogens, such as water or alcohols, with noadded catalysts. In this investigation, two differ-

Scheme 3. Synthesis of BACy1 and BACy2.


ent polymerization solvents were used because ofthe different solubility properties of the mono-mers. In particular, PAA-Cy1 was synthesized ina 6:1 (v/v) methanol/water mixture because of thepoor solubility of BACy1 in water, whereas PAA-

Cy2 was obtained from BACy2 in an aqueoussolution. High monomer concentrations give thebest results in PAA synthesis because the poly-merization reaction, a Michael-type addition, isan equilibrium reaction that is affected by the

Figure 1. 1H NMR spectrum of BACy1 run in CDCl3.

Figure 2. 13C NMR spectrum of BACy1 run in CDCl3.


reactant concentration and steric hindrance.Therefore, in this work, a 1.5 M concentration forall reagents was chosen. With carboxylated bisac-

rylamides as monomers, as in the case of BACy2,a stoichiometric amount of a base must be addedto the monomer mixture to prevent a reaction of

Figure 3. 1H NMR spectrum of BACy2 recorded in D2O.

Figure 4. 13C NMR spectrum of BACy2 recorded in D2O.


the acidic group with 2MP. In this case, lithiumhydroxide was used. The byproduct of acidifica-tion with HCl after the completion of the reaction,LiCl, was soluble in acetone and was dissolvedduring precipitation. The raw products were pu-rified by precipitation from water/acetone (ormethanol/acetone) mixtures after the solution pHwas reduced to 1–2 with 2 M HCl. Both PAA-Cy1and PAA-Cy2 were characterized with 1H and 13CNMR. The results were in full agreement with theproposed structures. The intrinsic viscosity ofPAA-Cy1, determined in a methanol solution, was0.15 dL/g, whereas that of PAA-Cy2, measured inwater, was 0.20 dL/g. The molecular weights, ob-tained by SEC operated in organic media for PAA-Cy1 and in aqueous media for PAA-Cy2, were15,000 and 10,000, respectively.

Kinetics of the Polymerization Reactions

In stepwise polymerization, the maximum attain-able number-average polymerization degree de-pends on the monomer conversion when otherconditions are the same. For this reason, we in-vestigated the polyaddition kinetics of 2MP withBACy1 and BACy2 in the selected solvents. Pur-posely designed kinetic experiments were carriedout in which the bisacrylamide consumption wasmonitored with 1H NMR spectroscopy. TheseNMR experiments were performed in a 6:1CD3OD/D2O mixture for BACy1 polymerizationand in D2O for BACy2. Lower reagent concentra-tions than those in conventional preparations (1.0M instead of 1.5–3 M) were chosen for these ki-netic studies to reduce solution viscosities and

Scheme 4. Synthesis of PAA-Cy1.

Scheme 5. Synthesis of PAA-Cy2.


this obtain better signal definition. The BACy1and BACy2 conversions were determined throughthe calculation of the reduction in the peaks dueto the resonance of the double-bond hydrogens at5.8 and 6.2 ppm, as described in the Experimentalsection. Typical changes in the 1H NMR spectraduring the polymerizations are shown in Figure 5for the BACy1/2MP polymerization.

Typical conversion curves for both PAA-Cy1and PAA-Cy2 experiments are shown in Figures 6and 7, respectively. These curves show that thepolymerization reaction of the BACy2 proceededmore quickly than that of BACy1. This could beascribed to the fact that the 1,4-unsaturated ad-dition of the amines to the acrylamides under-went general acid catalysis in the presence ofprotic solvents and water, which provided ahigher acid strength than methanol, and had asuperior catalytic effect on the BACy2 polymer-ization. An analysis of the experimental datademonstrated that, in both cases, the mechanismcorresponded to pseudo-second-order kinetics,with the kinetic constants including the solventconcentrations. The best fitting curves, obtainedwith eqs 1a and 1b, led to the kinetic constantskc,1, � 5.96 � 10�3 min�1 L mol�1 for BACy1/2MPpolymerization and kc,2 � 5.90 � 10�2 min�1 Lmol�1 for BACy2/2MP polymerization:

�d[BACy1]

dt � kc,1[BACy1][2MP] (1a)

�d[BACy2]

dt � kc,2[BACy2][2MP] (1b)

Degradation Studies

The hydrolytic degradation of these PAAs, which iscaused by the hydrolysis of the activated amidicgroups contained in the repeating units, has beenextensively investigated in our group under differ-ent hydrolytic conditions and found to depend onthe polymer structure and pH, with the times fordegradation into oligomeric products ranging from48 h to 2 months,24 according to the steric hindrancenext to the amidic group, the pH level, and theoverall molecule hydrophilicity. In this work, thehydrolytic degradation of water-soluble PAA-Cy2was investigated at pH 7.4 and 30 °C through themonitoring of the molecular weight reduction withtime via SEC analysis performed in an aqueousmedium. The results were comparable to those pre-viously observed for other amphoteric PAAs derivedthrough the polyaddition of different secondarybisamines to 2,2-bisacrylamidoacetic acid.15,22 Infact, PAA-Cy2 showed a relatively slow degradationrate in comparison with the rates of other nonam-photeric PAAs, with an 10–20% decrease in Mnafter 1 day and complete degradation only after 6–7days.

The reductive degradation of PAA-Cy2 was alsoinvestigated in a buffer solution at pH 7.4 and in thepresence of 2-mercaptoethanol as a reducing agent

Figure 5. 1H NMR spectra (25 °C, 400 MHz) of the reaction mixture composed of 1 MBACy1 and 1 M 2MP in 6:1 CD3OD/D2O after (a) 0, (b) 20, and (c) 46 h.


Figure 6. Polymerization reaction of BACy1 with 2MP (general conditions: solvent� 6:1 CD3OD/D2O, temperature � 25 °C, and [BACy1] � [2MP] � 1.0 M). The solid lineis the calculated curve, and the symbols represent experimental data.

Figure 7. Polymerization reaction of BACy2 with 2MP (general conditions: solvent� 6:1 CD3OD/D2O, temperature � 25 °C, and [BACy2] � [2MP] � 1.0 M). The solid lineis the calculated curve, and the symbols represent experimental data.


as a model for biological reducing environments (seeScheme 6). The results of typical degradation exper-iments, reported in Figure 8, show that after 1 day,

Mn underwent a 50% decrease, and degradationwas complete in almost 2 days. Because PAA-Cy1was not soluble in water, the reductive degradation

Scheme 6. Reductive degradation of PAA-Cy2 in the presence of 2-mercaptoethanol.

Figure 8. Reductive degradation of PAA-Cy2 in the presence of 2-mercaptoethanol ina TRIS buffer at pH 7.4 and 37 °C ([repeating unit of PAA-Cy2] � 0.02 M and[2-mercaptoethanol] � 0.10 M).


was investigated in organic solvents. Even underthese conditions, complete degradation was ob-served after less than 3 days.

CONCLUSIONS

The following general conclusions can be drawnfrom the results reported in this article:

1. Cationic and amphoteric PAA homopolymerscontaining a disulfide linkage in each repeat-ing unit, with different hydrophilic–hydro-phobic balances, were obtainable by the poly-addition reaction of 2MP in the presence ofBACy1 and BACy2, respectively.

2. The experimental conditions, including thechoice of protic solvent, mild temperatures,and no added catalysts, did not significantlydiffer from those normally used for conven-tional PAA synthesis. The molecularweights and molecular weight distributionswere also in the ranges usually observed forPAAs. Therefore, the adopted procedure canbe widely applied to the synthesis of copoly-meric PAAs containing different amounts ofdisulfide linkages in their main chains.

3. The disulfide-containing PAAs described inthis article and presumably all their copoly-mers with conventional PAAs may be, in prin-ciple, used for a number of purposes, includ-ing peptide modification and the attachmentof drugs, tracers, or other biologically activecompounds, and may also undergo reductivecleavage in biological environments, as dem-onstrated by model reactions carried out inthe presence of 2-mercaptoethanol.

4. The hydrolytic reaction of amphoteric PAA-Cys2 is not altered by the presence of thedisulfide bond and proceeds at a rate veryclose to that of model amphoteric PAAs.

5. Both PAA-Cys1 and PAA-Cys2, because oftheir potential as biomimetic and biodegrad-able polymers, are eligible to undergo fur-ther biological evaluation by purposely de-signed cytotoxicity and body distribution ex-periments. Preliminary in vitro tests25 haveindicated that the cytotoxicity of PAACy2 isprobably very low and comparable to that ofother amphoteric PAAs previously studiedby Duncan and coworkers at Cardiff Univer-sity (Wales, United Kingdom).15,18,23

This work was funded by the Italian MIUR (Ministeroper l’Universita e la Ricerca) progetto COFIN 2002.

REFERENCES AND NOTES

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New poly(amidoamine)s containing disulfide linkages in their main chain

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