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    Polymeric redox-responsive delivery systemsbearing ammonium salts cross-linked via disulfides

    Christian Dollendorf, Martin Hetzer and Helmut Ritter*

    Full Research Paper Open AccessAddress:Lehrstuhl fr Prparative Polymerchemie, Institut fr OrganischeChemie und Makromolekulare Chemie, Heinrich-Heine Universitt,Universittsstrae 1, Geb. 26.33.00, 40225 Dsseldorf, Germany

    Email:Helmut Ritter* - h.ritter@uni-duesseldorf.de

    * Corresponding author Equal contributors

    Keywords:cationic hydrogel; cross-linked polymer; 2-(dimethylamino)ethylmethacrylate (DMAEMA); disulfide cleavage; N,N-diethylacrylamide(DEAAm)

    Beilstein J. Org. Chem. 2013, 9, 16521662.doi:10.3762/bjoc.9.189

    Received: 26 April 2013Accepted: 24 July 2013Published: 13 August 2013

    Associate Editor: P. J. Skabara

    2013 Dollendorf et al; licensee Beilstein-Institut.License and terms: see end of document.

    AbstractA redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm) and 2-(dimeth-ylamino)ethyl methacrylate (DMAEMA). N,N-bis(4-chlorobutanoyl)cystamine was used as disulfide-containing cross-linker toform networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction ofdisulfide to mercaptanes by use of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or cysteamine, respectively. Thesoluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, meth-ylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recog-nized. Physical properties of the cross-linked materials, e.g., glass transition temperature (Tg), swelling behavior and cloud points(Tc) were investigated. Redox-responsive behavior was further analyzed by rheological measurements.


    IntroductionHydrogels with stimuli-responsive properties, in combinationwith targeted release of embedded drugs, have emerged as afascinating class of biomedical materials and attracted greatinterest in medical science, polymeric biotherapeutics and drugdelivery in general [1-4]. In this regard, poly(N-isopropylacry-lamide) (PNIPAM) and poly(N,N-dimethylamino)ethylmethacrylate (PDMAEMA) based hydrogels are the most

    extensively studied representatives. Due to the protonation ofcovalently attached tertiary amine groups, PDMAEMA exhibitspH-responsive behavior and is used in gene delivery [5-7]. Ingeneral, temperature- and pH-responsive hydrogels containinghydrophilic monomers show low toxicity along with a high effi-ciency in drug delivery [8]. Besides, cross-linked cationicnanogels have been suggested as DNA delivery systems [9-11].


  • Beilstein J. Org. Chem. 2013, 9, 16521662.


    Table 1: Results for the copolymerization of DEAAm and DMAEMA using different monomer ratios.


    conc.[wt %]

    AIBN[mol %]




    Mn,GPC[g mol1] PDI

    1:1 25 0.5 60 18 >99 37200 3.205:1 25 0.5 60 18 >99 71500 2.721:1 50 0.5 60 18 >99 57100 4.475:1 50 0.5 60 18 >99 121400 3.271:1 bulk 0.25 varyinga varyinga >99 187400 4.30

    a16 h at 60 C, 8 h at 70 C, 16 h at 80 C, and 3 h at 100 C.

    Hamamoto et al. reported a cross-linked polycationic hydrogelwhen investigating the swellingdeswelling behavior and theabsorbency of organic substances [12]. Positively charged poly-meric systems show an increased adsorption to negativelycharged cell membranes, a higher retention time within tumortissue [13-15] and the ability of DNA encapsulation used inanti-cancer treatment [16-23]. Further examples for polymericnetworks based on poly(ethylenimine) (PEI) or poly(diethylacrylamide) (PDEA) can be found in the literature [24-26].

    Disulfide bonds are generally stable in human blood circulationand extracellular milieus, but are prone to cleavage in a reduc-tive environment through dithioldisulfide exchange reactionsforming two corresponding thiol end-group bearing moieties[27,28]. The cleavage reaction is relatively fast and can takeplace within minutes to hours. This is considerably faster thanthe kinetics of other functionalities like esters and carbonates,whose degradation take days to weeks or even months insidethe human body [29-31]. Dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and glutathione tripeptide(-glutamyl-cysteinyl-glycine; GSH) are known as the mostcommon disulfide-cleaving agents [32,33]. Furthermore, GSHis the most abundant biological reductive agent in the humanbody with a 501000 fold excess in human cells compared toextracellular milieus. In tumor cells the GSH concentration iseven higher [34,35]. This large difference can be exploited forselective intracellular delivery and release of bioactive agentslike DNA or low molecular-weight drugs [34]. Especially in thehuman blood stream the molecular weight is of significantimportance for prolonged circulation cycles and enhancedselectivity for diffusion into tumor cells (enhancedpermeationretention effect) [13,36-41].

    Reductive cleavable polymeric networks can release embeddeddrugs within the reductive tumor-cell environment [34,42].There are several examples for micellar polymer systems,which bear disulfide functionalities and lose their structure aftera cleavage induced by the addition of DTT, and which subse-quently release the embedded substances, such as dyes or DNA

    molecules [33,43-45]. Other examples for disulfide cross-linkedh y d r o g e l n e t w o r k s a r e c o p o l y m e r s o f N - ( 2 -hydroxypropyl)methacrylamide (HPMA) and N-methacryloyl-glycylglycine, copolymers of DMAEMA and 1,4-bis(2-thioben-zoylthio)prop-2-yl)benzene, or cross-linked poly(L-lysine) [46-48].

    In the current work, we describe the synthesis of a novel redox-responsive polycationic hydrogel by cross-linking copolymersof N,N-diethylacrylamide (DEAAm) and DMAEMA with adisulfide-bearing cross-linking agent. The redox-responsivebehavior of the cross-linked poly(DEAAm-co-DMAEMA)hydrogel was shown by reversible enclosure and release ofdifferent dyes and further proven by rheological measurements.Additionally, physical properties of the cationic hydrogels, e.g.,glass transition temperature (Tg), swelling behavior, and cloudpoints (Tc) of the thiol-bearing copolymers after cleavage wereinvestigated. Although our results show only preliminaryinvestigations, this hydrogel system with its redox-responsivebehavior and polycationic structure could be a promisingsystem for medical applications.

    Results and DiscussionSynthesis of cross-linked hydrogels in solu-tion and inclusion-release investigations ofembedded dyesThe copolymerization of DEAAm and DMAEMA was carriedout in ethyl acetate as solvent using different concentrations(2550 wt %) and monomer ratios (1:1 and 5:1). Quantitativeconversions were reached. Solutions with higher concentrationsled to higher molecular weights during the reaction, but also tobroader weight distributions (refer to Table 1).

    The cross-linking agent N,N-bis(4-chlorobutanoyl)cystamine(CL 1) was synthesized by using the reaction of cystaminedihydrochloride and 4-chlorobutanoyl chloride [49]. Polycat-ionic hydrogels where obtained by adding CL 1 to a solution ofpoly(DEAAm-co-DMAEMA) and potassium iodide in acetone

  • Beilstein J. Org. Chem. 2013, 9, 16521662.


    Scheme 1: (a) Synthesis of a polycationic cross-linked hydrogel containing disulfide groups. (b) Reductive cleavage and oxidative rebuilding of disul-fide groups in the polymeric network using DTT and KOH/O2. (c) Enclosure and release of embedded dyes by reductive opening and oxidativerebuilding of the polymeric network.

    at 80 C. The polymeric network was formed via quaterniza-tion of the tertiary amine groups of DMAEMA with bothhalogen atoms of CL 1 (Scheme 1a). Potassium iodide wasadded to accelerate the quaternization by replacing the halogenatoms in a Finkelstein reaction. Using poly(DEAAm-co-DMAEMA) with a molar ratio of 1:1 (sample 3) resulted in abetter network formation, compared to a molar ratio of 5:1,because more tertiary amine groups are available for the

    halogen atoms of CL 1 to form cationic networks via quater-nization reactions. Additionally, the use of a DMAEMA/CL 1ratio of 2:1 results in a higher possibility of each tertiary aminegroups to react with halogen atoms to form quaternary ammoni-um groups. A high polymer concentration in the reaction mix-ture should lead to a higher network-density based on increasedintermolecular cross-linking in contrast to intramolecular quat-ernization reactions in diluted solution. After cross-linking of

  • Beilstein J. Org. Chem. 2013, 9, 16521662.


    Figure 1: Release of enclosed dyes from polycationic networks containing disulfide bonds after treatment with DTT.

    the copolymers by quaternization, the insoluble polycationichydrogels were filtered off and washed several times withacetone to remove unreacted CL 1 and potassium iodide.

    To determine the redox-responsive behavior of the cross-linkedpolymers, they were given into water with a subsequent addi-tion of DTT to dissolve the polymeric networks by the reduc-tion of the disulfide bonds to thiol side-chains (Scheme 1b). Thedissolved polymers were cross-linked again by the oxidation ofthe thiol groups to reform disulfide bonds using oxygen underbasic conditions at 60 C. As described above, it was crucial touse concentrated solutions, because only thiol groups ofdifferent polymeric chains in close proximi


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