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529 Full Paper Macromolecular Chemistry and Physics wileyonlinelibrary.com DOI: 10.1002/macp.201100564 Homologous Copolymerization Route to Functional and Biocompatible Polyvinylpyrrolidone Myriam G. Tardajos, Maria Nash, Yury Rochev, Helmut Reinecke, Carlos Elvira, Alberto Gallardo* Linear multifunctional polyvinylpyrrolidone (PVP) chains bearing carboxylate (COO ) or/and sulfonate (SO 3 ) groups are prepared by standard radical copolymerization of homologous vinylpyrrolidone (VP) and the VP derivatives 3-carboxyvi- nylpyrrolidone (VP C ) and 3-sulfoalkylvinylpyrrolidone (VP S ). Functional PVP networks with homogeneous crosslinking density are also prepared using a new homologous water- compatible VP-R-VP crosslinker. This homologous approach for the preparation of functionalized PVP systems is com- pared theoretically with the preparation of VP with commer- cial acrylic comonomers bearing similar functionalities. All the linear chains and networks are tested under basic in vitro cell cultures. polymers. [3] When adsorbed or anchored on surfaces, it resists the nonspecific adsorption of proteins. [4] Joined with iodine, it is used in the preparation of Betadine®. Addition- ally, it has been a classic component of contact lenses and is used in the food industry as additive, stabilizer (E1201), clarifying agent, etc. (approved for this use by the FDA [5] ). 1. Introduction Polyvinylpyrrolidone (PVP) is an amphiphilic, nonionic, biocompatible, and stable polymer, which is soluble in water and in many organic solvents. Its chemical structure is shown in Figure 1. It is a polymer with a large range of applications; today, it is certainly a first-choice polymer in the biomaterials area, and it was used as a plasma expander in World War II. [1] Furthermore, it was the polymer selected to prepare the first injectable polymer–drug conjugate. [2] In this area of bioconjugation, it is a very attractive support in cancer because of its high residence time in plasma and its low tissue distribution compared with other synthetic M. G. Tardajos, Dr. M. Nash, Dr. H. Reinecke, Dr. C. Elvira, Dr. A. Gallardo Instituto de Ciencia y Tecnología de Polímeros, ICTP-CSIC. Juan de la Cierva 3, 28006 Madrid, Spain E-mail: [email protected] Dr. Y. Rochev NCBES National Center for Biomedical Engineering Sciences National University of Ireland Galway, NUIG, Ireland Figure 1. Structure of the repeating unit of PVP. Macromol. Chem. Phys. 2012, 213, 529−538 © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Homologous Copolymerization Route to Functional and Biocompatible Polyvinylpyrrolidone

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Full PaperMacromolecularChemistry and Physics

Homologous Copolymerization Route to Functional and Biocompatible Polyvinylpyrrolidone

Myriam G. Tardajos, Maria Nash, Yury Rochev, Helmut Reinecke, Carlos Elvira, Alberto Gallardo*

Linear multifunctional polyvinylpyrrolidone (PVP) chains bearing carboxylate (COO − ) or/and sulfonate (SO 3 − ) groups are prepared by standard radical copolymerization of homologous vinylpyrrolidone (VP) and the VP derivatives 3-carboxyvi-nylpyrrolidone (VP C ) and 3-sulfoalkylvinylpyrrolidone (VP S ). Functional PVP networks with homogeneous crosslinking density are also prepared using a new homologous water-compatible VP-R-VP crosslinker. This homologous approach for the preparation of functionalized PVP systems is com-pared theoretically with the preparation of VP with commer-cial acrylic comonomers bearing similar functionalities. All the linear chains and networks are tested under basic in vitro cell cultures.

1. Introduction

Polyvinylpyrrolidone (PVP) is an amphiphilic, nonionic, biocompatible, and stable polymer, which is soluble in water and in many organic solvents. Its chemical structure is shown in Figure 1 . It is a polymer with a large range of applications; today, it is certainly a fi rst-choice polymer in the biomaterials area, and it was used as a plasma expander in World War II. [ 1 ] Furthermore, it was the polymer selected to prepare the fi rst injectable polymer–drug conjugate. [ 2 ] In this area of bioconjugation, it is a very attractive support in cancer because of its high residence time in plasma and its low tissue distribution compared with other synthetic

wileyon

M. G. Tardajos , Dr. M. Nash , Dr. H. Reinecke , Dr. C. Elvira , Dr. A. Gallardo Instituto de Ciencia y Tecnología de Polímeros, ICTP-CSIC. Juan de la Cierva 3, 28006 Madrid, Spain E-mail: [email protected] Dr. Y. Rochev NCBES National Center for Biomedical Engineering Sciences National University of Ireland Galway, NUIG, Ireland

Macromol. Chem. Phys. 2012, 213, 529−538© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

polymers. [ 3 ] When adsorbed or anchored on surfaces, it resists the nonspecifi c adsorption of proteins. [ 4 ] Joined with iodine, it is used in the preparation of Betadine®. Addition-ally, it has been a classic component of contact lenses and is used in the food industry as additive, stabilizer (E1201), clarifying agent, etc. (approved for this use by the FDA [ 5 ] ).

529linelibrary.com DOI: 10.1002/macp.201100564

Figure 1 . Structure of the repeating unit of PVP.

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In a similar way to polyethylenglycol (PEG)-based sys-tems, active groups, molecules or devices may be incor-porated into crosslinked PVP networks or into functional PVP chains by covalent attachment. This incorporation intends to transport those entities and/or to modulate their interaction with the aqueous medium, offering solubility in water or a “soft” and “charge-free” hydrated environment. In some cases, the center of gravity moves upon interaction with the environment from the active species to the polymeric nanotransporter. In this way, the interaction with other active molecules, solubility, bio-compatibility, unspecifi c protein adsorption, degradation, possible excretion, or recovery, etc. can be modulated. It has even been described that conjugates with antitumor agents can profi t from the imperfection of the newly formed tumor vasculature and extravasate in that area in a specifi c way [the so-called “enhanced permeation retention” (EPR) effect [ 6 ] ]. These properties, summed up to the intrinsic implications associated to its macro-molecular nature, make them attractive as biomedical and biotechnological utility.

However, PVP obtained in the conventional way (standard radical polymerization) does not possess func-tional groups. Many efforts are being undertaken to implement the alternative use of PVP in functionalization and conjugation. PVP chain ends can be functionalized during the polymerization of vinylpyrrolidone (VP), using, for example, functional transfer agents or specifi c chain capping agents in controlled (living) polymerization, to obtain chain ended conjugates, [ 7–12 ] structurally similar to those obtained with PEG. However, there is a great interest in the preparation of multi and polyfunctional lateral chain conjugates similar for instance to the derivatives of poly-hydoxypropylmethylacrylamide (PHPMA). There are

Figure 2 . Possible strategies for the functionalization of PVP in its sid

Macromol. Chem. Phys© 2012 WILEY-VCH Verlag Gm

different HPMA-doxorubicin conjugates on an advanced clinical stage. [ 13 ] In the case of PVP, this interest has for a long time been in confl ict with the chemical inertia of the lactam rings. There have been attempts to laterally func-tionalize PVP by modifying the polymer, hydrolyzing the ring, or modifying it with bromine at high temperatures (method A of Figure 2 ). [ 14–17 ] However, these reactions are diffi cult to control and are greatly inconvenient as it is impossible to purify the obtained products because all the units, including those that have suffered undesired sec-ondary reactions, are part of the common macromolecular chains. Because of the diffi culties in functionalizing PVP laterally by a controlled path, researchers have preferred to ignore polymer functionalization and carry out copoly-merizations (method B of the Figure 2 ) using commercial functional monomers such as maleic anhydride, acrylic acid, and so on, mainly to obtain covalent conjugates with different biologically signifi cant molecules. [ 18–20 ] This strategy, the most used, creates macromolecular chains that do not only contain VP units and it does not incorporate the functionality in the VP units but in the others. Besides, this kind of processes of copolymerization with heterologous functionalities implies the disadvan-tage related to the differential reactivities of the comono-mers that, in some cases may be very relevant, and lead to undesired compositional heterogeneities. [ 21 ] For these reasons, the best hypothetic alternative for the prepara-tion of PVP derivatives with poly- and multifunctional side chains with a versatile control of the number of func-tional groups and composition, and also with a random distribution of functional groups, is the modifi cation of the VP monomer followed by its homo- or copolymerization with unmodifi ed VP (method C of the Figure 2 ), that is, the homologous copolymerization route. As

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e chain.

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MacromolecularChemistry and Physics

N

Figure 3 . Structures of the VP derivatives used in this work.

functionalized VP derivatives are not commercially avail-able, the functionalized monomers or crosslinkers used in method C have to be synthesized. Several examples described by our group [ 22,23 ] and by others [ 24–29 ] can be found in the recent literature.

In this work, the feasibility of method C has been shown using two of the reported monomers with sulfonate [ 23 ] and carboxylate [ 27 ] groups [3-sulfoalkylvinylpyrrolidone (VP S ) and 3-carboxyvinylpyrrolidone (VP C ), see Figure 3 ]. Both functions COO − and SO 3 − have great interest for such a sol-uble and biocompatible carrier as PVP. The sulfonic group is found in heparin, a polysaccharide with a high biological signifi cance. Synthetic polymer bearing sulfonic groups have been proposed as heparin analogs or competitors in antithrombogenic [ 30 ] or antiangiogenic [ 31 ] applications, respectively. The carboxylic functions are preferred func-tionalities for activation–bioconjugation procedures. [ 32 ] It is shown here that, despite the mentioned synthetic effort needed to obtain the monomers, the polymerization is very simple, can be performed in water and allows a simple compositional tailoring and easy multifunctionality. Thus, the method is very facile and versatile. We have also faced the aspect of the differential reactivity in the radical copolymerization of VP comparing methods B and C theo-retically. The concepts described here are especially relevant in the formation of networks. It is shown that using a new water-soluble homologous crosslinker (VP -O- VP, see Figure 3 ) PVP hydrogels with tailored water uptake and function-ality can be easily prepared.

2. Experimental Section

2.1. Materials

Unless otherwise noted, materials were obtained from commer-cially available sources and used without purifi cation. The pure

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© 2012 WILEY-VCH Verlag Gmb

VP monomer was purifi ed by distillation at low pressure before use. The monomers VP C and VP S were prepared as described pre-viously by Bencini et al. [ 27 ] and by our group. [ 23 ]

2.2. Synthesis of VP-O-VP

A freshly distilled solution of VP (5 mL, 46.8 mmol, 4 equiv.) in anhydrous tetrahydrofuran (THF, 30 mL) was added dropwise to a commercial solution of lithium diisopropylamide (2.0 M , in THF, hexane, and ethylbenzene, 26 mL, 4 equiv) in inert atmos-phere and at − 78 ° C and stirred for 2 h. Then, 2-bromoethyl ether (11.7 mmol, 1.0 equiv) was added dropwise. The resulting solu-tion was magnetically stirred for 2 h at − 78 ° C and then at room temperature over night. At the end of the reaction, monitored by thin-layer chromatography (TLC), the solution was hydrolyzed in CH 2 Cl 2 /H 2 O (2:1, 300 mL). The aqueous layer was extracted with CH 2 Cl 2 (2 × 100 mL), and the organic layers were combined and dried over sodium sulfate (Na 2 SO 4 ) and the solvent evaporated at low pressure. The residue was purifi ed by column chromatog-raphy using CH 2 Cl 2 /diethyl ether (20:1) as eluent. 1 H NMR (CDCl 3 , δ ): 7.09 (dd, 2H, N–C H = , J = 16.0 and 8.9 Hz), 4.41 (m, 4H,N–CH = C HH), 3.49 (m, 8H, N–C H 2 and O–C H 2 ), 2.63 (m, 2H, CO–C H) , 2.31,2.16 (m, 4H, N–CH 2– C H 2 ), 1.77,1.62 (m, 4H, O–CH 2 –C H 2 ) . MS (Na + ): m / z = 293 (MH + ) ESI-HRMS for C 16 H 24 N 2 O 3 (MH + ): 293.1792, Found: 293.1917 .

2.3. Polymerizations

2.3.1. Linear polymers

Monomers and initiator azoisobutyronitrile (AIBN) were dis-solved in water in a total concentration of 1 mol L − 1 and 1.5 × 10 − 2 mol L − 1 respectively. Nitrogen was fl ushed through the polymerizing solution for 30 min. Then polymerization was started at the chosen temperature. The formed polymer or copoly mer was isolated and purifi ed by dialysis in membranes of cut-off 3000 followed by freeze-drying.

The analysis of the composition was carried out by comparison of the integrated intensities of resonance signals. For VP S resonance signals with chemical shifts at δ = 2.77 (2H VPs ), 3.36 (2H VP + 2H VPs ), 3.5 (H VP + H VPs ) were used. For VP C resonance signals with chemical shifts below δ = 2.8 (6H VP + 4H VPc ) and above δ = 2.8 (3H VP + 4H VPc ) were used.

2.3.2. Networks

The hydrogels were prepared by photopolymerization with Ciba-Geigy Irgacure 369 as initiator using the novel crosslinker VP-O-VP. These networks were synthesized by UV-initiated free radical crosslinking polymerization. VP, crosslinker (1 or 10 mol%), ini-tiator (0.5%, w/w), and the desired percentage of functionalized VP were mixed. Water was added if needed to solubilize VP S . Nitrogen was fl ushed through the polymerizing solution for 10 min. The solution was placed in a polypropylene holder and covered with another polypropylene holder in order to avoid air contact during the polymerization. Polymerization was facili-tated via irradiation with UV light at 365 nm for 10 min with a Mercury Lamp Osram HQL (250 W). Afterward the networks were

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Table 1. VP S and VP C molar fractions in the feed ( F VPs and F VPc ) and in the copolymers ( f VPs and f VPc ) for the statistical copoly-merization reactions carried out at low conversion.

F VPs or F VPc f VPs f VPc

0.2 0.16 0.19

0.4 0.33 0.31

0.6 0.49 0.55

0.8 0.79 0.8

removed from the holder and immersed in distilled water for at least 3 d to remove any unreacted chemicals. During this time, the water was replaced several times.

2.4. Determination of Reactivity Ratios

Assuming that the radical copolymerization is governed by the terminal model, [ 33 ] the reactivity ratios of the copolymerization of VP S or VP C and VP in water were determined using the soft-ware Copol. [ 34 ] The terminal model considers that the reactivity of the growing chain depends on the nature of the terminal unit and needs two parameters — in the case of binary copolymeriza-tions — named reactivity ratios to describe the reaction. These reactivity ratios are given by r 1 = k 11 / k 12 and r 2 = k 22 / k 21 , where k ij ( i,j = 1,2) is the rate constant for the addition of a monomer M j to a propagating chain end M i . A reactivity ratio r 1 higher or lower than 1, means that a growing chain end M 1 is more reac-tive toward monomer 1 or 2, respectively.

The software Copol uses compositional data of copolymers studied over a wide composition interval with molar fractions in the monomer feed, from 0.20 to 0.80 as it is shown in Table 1 , and determines the reactivity ratios according to the general copolymerization equation by application of the nonlinear least-squares treatment proposed by Tidwell and Mortimer. [ 35 ] The copolymers were prepared in the same conditions as those described in the previous section, and the reaction time was adjusted to obtain conversions lower than 5 wt% to satisfy the copolymerization equation. [ 36 ]

The molar fraction of monomer units in the copolymer chains was determined from the 1 H NMR spectra of the copolymer samples prepared with different monomer feed. The analysis was carried out by comparison of the integrated intensities of resonance signals. For VP S resonance signals with chemical shifts at δ = 2.77 (2H VPs ), 3.36 (2H VP + 2H VPs ), 3.5 (H VP + H VPs ) were used. Reactivity ratios values of r VP = 1.61 and r VPs = 0.90 have been obtained. For VP C resonance signals with chemical shifts below δ = 2.8 (6H VP + 4H VPc ) and above δ = 2.8 (3H VP + 4H VPc ) were used. Reactivity ratios values of r VP = 1.50 and r VPc = 0.99 have been obtained.

2.5. Methods

Structural characterization of the polymers was carried out by NMR. 1 H NMR analysis of the copolymers was performed using a Bruker Avance-300 spectrometer (300 and 75.4 MHz, respectively) in D 2 O with tetramethylsilane (TMS) as the internal standard. The chemical shifts are given in the δ scale relative to TMS. Poly mer

Macromol. Chem. Phys.© 2012 WILEY-VCH Verlag Gm

compositions were determined from the 1 H NMR spectra by the integrated intensities of proton NMR units, as described in the previous sections.

Mass spectra were recorded on an HP series 1100 MSD spectrometer and elemental analysis on a Heraeus CHN–O Rapid analyzer.

The molecular weight was analyzed by size-exclusion chromatography (SEC, Shimadzu SIL 20A-HT) with an isocratic pump (serial LC-20D) connected to a differential refractometric detector (serial RID-10A). Three columns of PL-aquarel OH 50, 40, and 30 (Polymer Laboratories; 8 μ m) were conditioned at 40 ° C and used to elute the samples (1 mg mL − 1 concentration) with mobile phase 0.2 M NaNO 3 , 0.01 M NaH 2 PO 4 buffered solution at pH 9 at 1 mL min − 1 . Calibration of SEC was carried out with monodisperse poly(ethylene glycol) standards in the range of 1.0 × 10 3 to 500 × 10 3 , obtained from Scharlab.

The water uptake was determined from thermogravimetric analysis (TGA) with a TA TGAQ500 (10 ° C min − 1 under 20 mL min − 1 dry nitrogen).

Photopolymerizations were carried on with a Mercury Lamp Osram HQL (250 W).

2.5.1. In vitro Biocompatibility Studies

The biocompatibility of the linear copolymers and networks was tested in vitro using Swiss 3T3 fi broblast cells. 3T3 cells were cultivated in Dulbecco's modifi ed Eagle medium (DMEM), sup-plemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin antibiotics, and maintained in a humidifi ed incu-bator at 37 ° C and 5% CO 2 . The cells were sub-cultured every 3 d. When cells reached 80–90% confl uence, cells were harvested and used for reseeding or for experimentation where appropriate. For experimentation, cells were seeded in triplicate at a density of 2 × 10 4 cells mL − 1 .

For the linear copolymers, cells were seeded as described above in sterile 24-well tissue culture plastic (TCP) plates and incubated for 24 h. After this time, the cell culture medium was removed and replaced by fresh medium, which contained polymers in the following concentrations 0, 1, 10, 100, 1000 μ g mL − 1 , and reincubated for 24 h. After this time, dsDNA Picogreen assay was carried out to assess the comparative biocompatibility of the prepared linear copolymers. As PVP is well known to be highly biocompatible it was used as the positive control.

DNA was measured using the dsDNA picogreen kit (Quant-iT™ PicoGreen® dsDNA Assay Kit) according to the manufacturer’s protocol. Briefl y, 100 μ L of the cell samples that have been frozen and defrosted repeatedly to induce cell lysis was added to a 96-well plate and a calibration curve was prepared with the DNA standards and TE/tritonX-100 buffer. About 100 μ L PicoGreen dye was added to each of the 96-well and incubated in the dark at room temperature for 5 min. Samples and standard curve were read on a fl uorescence plate reader (Perkin-Elmer Victor 3) at 485/535 nm (excitation/emission).

For the networks, cells were seeded in the presence of the hydrogels in complete medium in a sterile 24-well culture plate and incubated for 48 h. After this time, all samples were checked under a microscope and the amount of living cells were visually assessed.

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Figure 5 . Theoretical instantaneous VP molar fraction in the copolymer as a function of the conversion for three reactions with initial VP feed molar fractions of 0.75, 0.50, and 0.25. Solid and dashed lines correspond respectively to the systems with sul-fopropyl methacrylate and methacrylic acid as comonomers.

0.0 0.2 0.4 0.6 0.80.0

0.2

0.4

0.6

0.8

1.0

FVP

=0.25

FVP

=0.50

FVP

=0.75

f VP-c

opol

ymer

conversion

3. Results and Discussion

When trying to derivatize PVP by using method B of Figure 2 and choosing commercial monomers as methacrylics, the differential reactivity issue should be considered. VP is a particular monomer that, in copolymerization with standard methacrylics, has a much lower reactivity than the methacrylics toward growing radicals mainly due to the higher polarizability of the double bond. Typical VP reactivity ratio values are below 0.1 while the comonomer parameter ranges from values close to 1 to much higher than 1. [ 37 ] This means that the growing macroradicals ended in VP are much more reactive to the methacrylic units than to VP. As a consequence, it is unavoidable that polymerizations carried out under non-continuous condi-tions have a high compositional heterogeneity. Homoge-neous products can be obtained when VP is copolymerized with homologous VP derivatives according to method C in Figure 2 .

Thus, to achieve the proposed systems bearing COO − or/and SO 3 − groups by radical copolymerization with pure VP, method B and commercial monomers such as sulfo-propyl methacrylate (M S ) and methacrylic acid (M C ) can be used respectively. As has been mentioned before, using method B, the PVP backbone integrity is lost because methacrylic units are incorporated. Therefore, at high comonomer loads, it is questionable to name the systems as PVP derivatives (see Figure 4 ).

The differential reactivities of the species in copolymer-ization should cause a major concern. Using the software Copol [ 34 ] and the reactivity ratios indicated in Figure 4 , which have been taken from literature, [ 37 , 38 ] the copoly-merization in a closed vessel can be described composi-tionally as a function of the conversion (Figure 5 ). There is a “dramatic” compositional heterogeneity, which is related to the differences in reactivities. As the meth-acrylics are much more reactive, the reaction begins

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Figure 4 . Scheme of the tentative functionalization of PVP by copol

N

O

O

O

-O3S

rVP=0.06

rMs=6.6

HO

O

radical

copolymerization

rVP=0.07

rMc=4.7

N

O

radical

copolymerization

O

O

Macromol. Chem. Phys© 2012 WILEY-VCH Verlag Gm

forming chains much richer in methacrylate than the nominal values of the initial feed composition. When these methacrylics are being consumed, chains rich in VP are formed. This issue is especially relevant at low methacrylic compositions (indicated in the example F VP = 0.75), which is very interesting since the copoly-mers should be most similar to native PVP. At this com-position, a continuous gradient from f VP = 0.3 till 1 is formed at conversions lower than 0.6. If the target is to prepare functionalized PVP with defined and con-trolled function loads, these copolymerizations are just unacceptable.

A particular case is the copolymerization of VP with non-homopolymerizable monomers such as maleates (or fumarates), which have been extensively used in the

533

ymerization with commercial methacrylics.

HOOC

COOH

-O3S

SO3-

N

HO

O

nm

N

O

O

nm

-O3S

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Figure 6 . Scheme of the alternating copolymerization of VP and dimethylfumarate .

literature to obtain so-called functional PVP. [ 18–20 ] The PVP with carboxylic groups addressed before, can be obtained easily in this case by hydrolysis of the maleic anhydride or ester. The mentioned differential reactivity of VP is still the same and macroradicals ended in VP are much more reactive toward maleic derivatives. However the macro-radical end in the acrylic unit must react with the less reactive VP since it cannot homopolymerize (or it is very diffi cult) because of the well-known steric hindrance of 1,2-disubstituted acrylics. As a consequence, almost pure alternating sequences are formed until one of the mono-mers is consumed (see Figure 6 ). Despite the utility of these alternating copolymers, it should be noted that it is not possible to modulate the functional load because as only alternating sequences are formed there is only one composition available, the equimolar one. Besides, this macromolecule cannot be described as a functionalized PVP since the integrity of the backbone is lost, that is, the macromolecule is not PVP anymore but another type of chain.

The scenario is completely different if homologous VP derivatives are used (see Figure 7 ). The structural and elec-tronic homology strongly reduces the differential reac-tivity in copolymerization. The reactivity ratios indicated

4

Figure 7 . Scheme of the functionalization of PVP by copolymerization

N

O

rVP=1.61

rVPS=0.9

radical

copolymerization

N

O

radical

copolymerization

rVP=1.50

rVPC=0.99

N

O

NaOOC

N

SO3Li

O

O

O

Macromol. Chem. Phys.© 2012 WILEY-VCH Verlag Gm

in Figure 7 have been calculated as described in Section 2 for the reactions in water.

The reactivity ratios are much closer to unity than in the previous examples, what is in agreement with a more similar reactivity in the polymerization process. As a consequence, copolymers obtained at high conversion are much more homogeneous than those obtained in the reactions with commercial methacrylates (see Figure 8 ). In this case, defi ned and quite homogeneous materials can be obtained when polymerizing in a closed vessel. The probability to obtain at high conversions an average composition similar to the nominal value used in the feed is very high. This holds even in the case of copolymers with low functionality. Thus by using functionalized VP, it is possible to produce homogeneous functionalized PVP with load control and true PVP backbone, what is not pos-sible when copolymerizing with commercial methacrylics or maleic derivatives.

Furthermore, the polymerization procedure is very simple and can be carried out in water. The appropriate amount of comonomers (according to the desired func-tional load) is mixed in water and allowed to polymerize under standard radical conditions, which means for instance adding initiator, bubbling nitrogen, (displacing

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with homologous VP derivatives.

HOOC

COOH

-O3S

SO3-

N

N

O

NaOOC

nm

N

N

O

nm

SO3Li

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Figure 8 . Theoretical instantaneous VP molar fraction in the copolymer as a function of the conversion for three reactions with initial VP feed molar fractions of 0.75, 0.50, and 0.25. Solid and dashed lines correspond respectively to the systems with sulfopropyl-VP (VP S ) and carboxy-VP (VP C ) as comonomers.

0.0 0.2 0.4 0.6 0.80.0

0.2

0.4

0.6

0.8

1.0

FVP

=0.25

FVP

=0.50

FVP

=0.75

f VP

-cop

olym

er

conversion

Figure 9 . Structure of the linear copolymers.

oxygen) and heating to 60 ° C. To show the feasibility and versatility of this method, the copolymers quoted in Table 2 have been prepared, varying the load of both functions and also preparing multifunctional terpolymers (entries 10 and 11 of Table 2 ). The generic structure is shown in Figure 9 . The polymers have been purifi ed and isolated by dialysis and freeze-drying. NMR analyses of polymers obtained at high conversions are in agreement with the initial feed compositions. As will be seen, Picogreen anal-ysis has shown that all copolymers are cytocompatible.

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Table 2. Initial and experimental composition of the copolymers.

Copolymer Feed

F VP F VPs

P1 poly(VP) 1 0

P2 poly(VP- stat -VP S ) 75:25 0.75 0.25

P3 poly(VP- stat -VP S ) 50:50 0.50 0.50

P4 poly(VP- stat -VP S ) 25:75 0.25 0.75

P5 poly(VP S ) 0 1

P6 poly(VP- stat -VP C ) 75:25 0.75 0

P7 poly(VP- stat -VP C ) 50:50 0.50 0

P8 poly(VP- stat -VP C ) 25:75 0.25 0

P9 poly(VP C ) 0 0

P10 poly(VP- stat -VP S - stat -VP C ) 25:25:50

0.25 0.25

P11 poly(VP- stat -VP S - stat -VP C ) 25:50:25

0.25 0.50

a ) Obtained from NMR analysis; b ) Obtained from SEC analysis, PI = poly

Macromol. Chem. Phys. © 2012 WILEY-VCH Verlag Gm

3.1. Networks

The previous discussion on reactivities can be extended to the process of PVP network formation by adding a small amount of difunctional crosslinker during the polymeriza-tion reaction. In this case, the compositional heterogeneity of VP and crosslinker implies also a structural heteroge-neity (in crosslinking density), which is a critical para-meter in a network. [ 39 ]

One of the most used commercial crosslinkers is eth-ylene glycol dimethacrylate ( EGDMA, see Figure 10 ). A copolymerization of VP with EGDMA at low EGDMA ini-tial feed molar fractions (1, 5, and 10 mol%) will lead to the reactions described by Figure 10 (using the reactivity ratios of the example VP/sulfopropyl methacrylate). As the

535

Copolymer

F VPc f VPs a ) f VPc a ) 10 3 Mn /PI b )

0 0 – 111/3.22

0 0.26 – 65/3.06

0 0.48 – 32/1.64

0 0.75 – 34/1.67

0 1 – 23/1.88

0.25 – 0.21 96/2.35

0.50 – 0.47 100/2.26

0.75 – 0.72 77/3.40

1 – 1 51/1.76

0.50 0.21 0.45 40/2.03

0.25 0.47 0.23 54/2.22

dispersity index.

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0,0 0,2 0,4 0,6 0,80,0

0,2

0,4

0,6

0,8

1,0

O

O

O

O

Of cros

s-cop

olym

er

conversion

Fcross

=0.1

Fcross

=0.05

Fcross

=0.01

Figure 10 . Theoretical instantaneous methacrylate molar frac-tion in the copolymer as a function of the conversion for three reactions with initial crosslinker feed molar fractions of 0.1, 0.05, and 0.01.

Figure 11 . Theoretical instantaneous VP/crosslinker molar frac-tion in the copolymer as a function of the conversion for three reactions with initial crosslinker feed molar fractions of 0.1, 0.05, and 0.01.

0,0 0,2 0,4 0,6 0,80,0

0,2

0,4

0,6

0,8

1,0N

O

O

NO

f cros

s-cop

olym

er

conversion

Fcross

=0.1

Fcross

=0.05

Fcross

=0.01

N

O

O

NO

N

SO3-

O

N

O

nm

r

Figure 12 . General chemical structure of the networks.

crosslinker is commonly used in low ratio, the reactions belong to the most problematic interval according to our previous discussion: the interval with low functionality. A gradient that depends on the crosslinker molar fraction is obtained during the fi rst stages of the reaction until the methacrylate is mostly consumed. As mentioned above, this compositional heterogeneity is related to a structural inhomogeneity in crosslinking density. Up to a certain conversion, a crosslinking gradient is obtained and after crosslinker consumption, new chains do not crosslink and poor quality network formation takes place. In practice, crosslinked PVP is not usually prepared by polymerization in the presence of a crosslinker as seen here but rather by crosslinking of preformed linear chains of PVP by dif-ferent methods as ionizing radiation. [ 40 ]

Figure 11 shows the reaction with the homologous crosslinker (using the reactivity ratios of the example VP/sulfopropyl-VP). The network is clearly superior to that of the previous example seen in Figure 10 , as the

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Table 3. Initial and experimental composition of the copolymers.

Network

Feed Network

F cross F VPs f cross a ) f VPs a ) Water uptake b ) [g H 2 O/ g polymer]

N1 cross poly(VP) 0.05 0 0.04 2.50

N2 cross poly(VP) 0.10 0 0.10 0.90

N3 cross poly(VP- stat -VP S ) 95:05 0.10 0.05 – 0.05 0.88

N4 cross poly(VP- stat -VP S ) 90:10 0.10 0.10 – 0.10 1.04

N5 cross poly(VP- stat -VP S ) 80:20 0.10 0.20 – 0.18 1.06

a ) Obtained from elemental analysis; b ) Obtained from TGA analysis.

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Figure 13 . Amount of DNA equivalent to the number of living cells measured on copolymers with different amounts of VP s using PVP as a reference.

Figure 15 . 3T3 fi broblast cells grown in the presence of one of the synthesized hydrogels. Scan bar size 200 μ m.

Figure 14 . Amount of DNA equivalent to the number of living cells measured on copolymers with different amounts of VP c using PVP as a reference.

Macromol. Chem. Phys. 2012, 213, 529−538© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, We

former is characterized by a homoge-neous crosslinking throughout the whole reaction.

The process of network formation is quite simple and networks with tailored crosslinking density and tailored amount of functional groups can be obtained by simul-taneously polymerizing the appropriate amount of crosslinker and monomers. To show this simplicity and versatility, we have prepared the networks quoted in Table 3 with different crosslinking degrees and sulfonic group loads. Figure 12 shows a generic structure of the net-works. In this case, photopolymerization, which is the preferred technique for the preparation of networks and crosslinked membranes, was used. A new water-soluble crosslinker was prepared for this purpose because the crosslinker reported in literature [ 24 ] is non-soluble in water.

Entries N1 and N2 show that the water uptake of the hydrogel can be modulated by the crosslinking density. It is also inter-esting that the water uptake in N3, N4, and N5 is very similar to that in N2, which indi-cated that they are all in their maximum expansion.

3.2. Biocompatibility Assay

The comparative biocompatibility of linear copolymers (P1–P9) of different concen-trations was assessed using the Picogreen assay. The assay results shown in Figure 13 and Figure 14 indicate that all copolymers

in the concentration range of 1–1000 μ g mL − 1 were highly biocompatible, with recovered DNA quantities similar to those recovered from the positive PVP controls.

Finally, the biocompatibility of the synthesized pure VP networks (N1, N2, N3, N4, and N5) was assessed by seeding 3T3 fi broblast cells and incubating for 48 h and observing visually. As can be seen in Figure 15 , cells attached and proliferated in the presence of the hydrogel on TCP. It can also be seen that no cells adhere to the hydrogel because of the highly hydrophilic nature of the chemical structure.

4. Conclusion

Multifunctional linear water-soluble PVP chains bearing car-boxylate COO − or/and sulfonate SO 3 − groups can be prepared very easily in water by the homologous copolymerization

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route using VP derivatives. Functional PVP networks with homogeneous crosslinking density can be prepared using a new homologous water-soluble crosslinker. It has been theoretically shown here that this homologous approach for the preparation of functionalized PVP systems is supe-rior to the copolymerization of VP with commercial acrylic comonomers bearing similar functionalities. The reasons for this superiority are: (1) integrity in the PVP backbone, (2) homogeneous copolymer composition (or crosslinking density in the case of networks) and (3) easy control of the functionality load just by comonomer ratio adjustment. All the linear chains and networks have shown to be biocom-patible under basic in vitro cell culture testing.

Acknowledgements : The authors gratefully acknowledge support from MAT 2007-63355 and MAT2010-20010, and from a FPI grant from the Ministerio de Ciencia e Innovación.

Received: October 11, 2011; Published online: February 17, 2012; DOI: 10.1002/macp.201100564

Keywords: biocompatibility; functionalization; monomer reacti-vity; networks , polyvinylpyrrolidone

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