The Role of Charge Density and Hydrophobicity on the Biocidal Properties of Self-Protonable Polymeric Materials.

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The Role of Charge Density and Hydrophobicity on the Biocidal Properties of Self-Protonable Polymeric Materials Simona Matrella, Carmela Vitiello, Massimo Mella, Giovanni Vigliotta, Lorella Izzo* Intrinsic antimicrobial thermoplastic A(BC)n copolymers (n ¼ 1, 2, 4), where A was poly(ethylene glycol) (PEG), BC was a random chain of methylmethacrylate (MMA), and alkyl-aminoethyl methacrylate (AAEMA), were synthesized and the antimicrobial activity and hemolyticity were evaluated on plaques obtained by casting as a function of the architecture, the N-substituent groups of the AAEMAs (methyl, ethyl, isopropyl, and tert-butyl groups) and the hydrophobic/charge density balance. Antimicrobial effectiveness and efficiency is controlled by the surface charge density and by the influence of N-alkyl groups on the surface morphology. Also interestingly, it is the absence of hemolitytic activity in all copolymers. In presence of Escherichia coli, the A(BC)2 copolymer with 40% of N-methyl groups is the most efficient, killing 91% of the bacteria already after 1.5 h.

Non-leaching, antimicrobial surfaces can be prepared using two synthetic approaches: the ‘‘grafting onto’’ technique,

S. Matrella, C. Vitiello, G. Vigliotta, L. Izzo degli Studi di Dipartimento di Chimica e Biologia, Universita Salerno, via Giovanni Paolo II, 132, Fisciano SA I-84084, Italy M. Mella degli Studi Dipartimento di Scienza ed Alta Tecnologia, Universita dell’Insubria, via Valleggio, 9, Como CO I-22100, Italy G. Vigliotta, L. Izzo NANOMATES, Research Centre for Nanomaterials and Nanotechnology, via Giovanni Paolo II, 132, Fisciano SA I-84084, Italy E-mail: [email protected] Supporting Information is available online from the Wiley Online Library or from the author. L.I., G.V., and M.M. are senior authors of this manuscript. a

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

with antimicrobial polymers generally containing quaternary ammonium groups (QA) being covalently attached onto the surfaces, or the ‘‘grafting from’’ technique, where the polymerization is carried out from chemically modified surfaces.[1] Although these techniques can be used to coat structural materials such as glass,[2–5] polymers,[6–13] paper,[5] and metals,[14] they both present advantages and disadvantages. For instance, the former is more suitable for preparing polymeric coatings with architecturally complex polymers at the cost of sacrificing the uniformity of the grafting density. The ‘‘grafting from’’ technique, on the contrary, can provide greater control of the grafting density, but hinders the preparation of polymeric structures (e.g., branched) that can potentially improve surface antibacterial activity. In both cases, the QA groups are introduced with the quaternization of amino-groups of the monomers or of the polymer chain. To improve on a few of the mentioned issues, three of us recently developed a new family of non-leaching, antimicrobial copolymers based on non-quaternized

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Macromol. Biosci. 2015, DOI: 10.1002/mabi.201400503

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1. Introduction

Early View Publication; these are NOT the final page numbers, use DOI for citation !!

S. Matrella, C. Vitiello, M. Mella, G. Vigliotta, L. Izzo www.mbs-journal.de

amino-groups and having an A(BC)n (n ¼ 1, 2 or 4) architecture. Such materials can be considered as the first example of intrinsically bactericidal polymers.[15] The copolymers were rationally designed to be nonsoluble in water and to possess bactericidal properties. Poly(ethylene glycol) monomethylether (mPEG, ‘‘A’’) was introduced to inhibit protein adhesion and to foster the hydrophilic environment needed for the build up of charge via water protonation. In this way, it was deemed possible to avoid the quaternization of amino groups. Methylmethacrylate (MMA, ‘‘B’’) was used as spacer for 2dimethylaminoethyl methacrylate (DMAEMA, ‘‘C’’) and to provide water-insoluble copolymers. Finally, ‘‘C’’ was the potentially antimicrobial monomer, as it bears a waterprotonable pedant amino-group. Indeed, experimental results showed that plaques obtained by casting A(BC)n copolymers were antimicrobial. A(BC)2 and A(BC)4 possessed a marked bactericidal activity toward both Gram positive and negative bacteria; plaques produced with linear copolymer A(BC) were substantially less active. The bactericidal activity demonstrated that quaternization of amino-groups was not needed to generate a surface charge density sufficiently high to efficiently kill bacteria, at least with the branched architectures. We rationalized this as due to the higher probability of forming strong hydrogen bonds of the type (–N(R1)(R2). . .HþN(R1) (R2)–) in the latter cases (also, vide infra Scheme 1). A mechanism by contact involving the leaching of divalent cations from bacteria cell wall was proposed, as neither the cationic pedant groups nor the polymer arms were long enough to completely penetrate the cell wall reaching the cytoplasm. Apart from our experimental findings, it is worth noting that there are no mechanistic studies in the literature that may foster the rational design of efficient antimicrobial non-leaching and non-toxic thermoplastic polymers, while design criteria are generally available to generate antibacterial polymer-coated surfaces.[16–19] In this report, we would like to give a contribution in filling this gap by studying the relationship between the antimicrobial/ hemolytic activity and hydrophobicity/charge density of plaques made of A(BC)n-type copolymers. With respect to composition, the hydrophobicity/cationic charge balance can be varied using different approaches. When the ‘‘segregated monomer’’ approach is applied to random copolymers of non-polar and cationic

monomers,[20] the balance is adjusted either changing the hydrophobic moieties or the ratio between the two monomers. In the ‘‘facially amphiphilic’’ approach, nonpolar and cationic parts are contained within the same monomer, and the hydrophobic/hydrophilic balance is varied by changing the hydrophobic group.[20] Finally, the ‘‘same centered’’ approach consists in obtaining homopolymers where hydrophobic groups are directly attached to a positively charged group.[21] To collect as much information as possible, the present study exploits both the ‘‘same centered’’ and the ’’segregated’’ approaches preparing A(BC)n copolymers where the N-alkyl substituents of alkyl-aminoethyl methacrylates (AAEMAs) and the AAEMA/MMA ratio were varied. It is important to notice that any variation in the N-substituent groups (methyl, Me; ethyl, Et; isopropyl, i-Pr; and tert-butyl, t-Bu) may also affect the surface charge density through a change in the pKb of the amines, and in their propensity to form strong hydrogen bonds stabilizing the proton charge. In this way, we de facto gain an additional handle to finetune the charge density, which may hopefully help in shedding some light on the role played by the mentioned parameters in influencing both antimicrobial and hemolytic activities.

2. Experimental Section 2.1. Materials Poly(ethylene glycol) monomethylether (mPEG) (Mn ¼ 2 000 Da, benzaldehyde dimethyl acetal, 2,2-bisMw/Mn ¼ 1.16), (hydroxymethyl) propionic acid, p-toluenesulfonic acid monohydrate (TsOH), acetone, N,N0 -dicyclohexylcarbodiimide (DCC), 4(dimethylamino) pyridine (DMAP), methanol, Pd/C 10%, 2-bromoisobutyryl bromide (BMPB), triethylamine (TEA), diethyl ether, ethanol, CuBr, 2,20 -bipyridine (bpy), chloroform, fluorescein sodium salt, cetyltrimethylammonium chloride (25 wt.-%) and Al2O3 were purchased from Aldrich and used without further purification. All manipulations involving air-sensitive compounds were carried out under nitrogen atmosphere using Schlenk or drybox techniques. Toluene (Aldrich) was dried over sodium and distilled before use. CH2Cl2 (Carlo Erba) was dried over CaH2 and then distilled. Methyl methacrylate (MMA), and the 2-(alkylamino) ethyl methacrylate (AAEMA) monomers such as 2-(dimethylamino) ethyl methacrylate (DMAEMA), 2-(diethylamino) ethyl methacrylate (DEAEMA), 2-(diisopropylamino) ethyl methacrylate (DIPAEMA), 2-(tert-butylamino) ethyl methacrylate (TBAEMA)

Scheme 1. Reaction for the formation of strong hydrogen bond between protonated and neutral AAEMA pendants.

Macromol. Biosci. 2015, DOI: 10.1002/mabi.201400503 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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(Aldrich) were dried over CaH2 and then distilled under reduced pressure of nitrogen. E. coli (strain JM109) were purchased from Promega (http:// www.promega.com/products; cat. No P 9751).

(–OCH3, MMA), 54.5 (CH2 main chain), 57.2 (–O–CH2–CH2–N(CH3)2), 63.2 (–O–CH2–CH2–N(CH3)2), 70.7 (–OCH2CH2–), 176.3–178.2 (–C5 5O). The molar mass of copolymers was determined by the degree of polymerization (DP) as evaluated from 13C NMR, by the molar mass of monomers (MM) and by the signal intensities (I)

2.2. Synthesis of mPEG-(PMMA-PAAEMA)n Copolymers 2.2.1. Synthesis of mPEG-Br Linear, mPEG-Br2, and mPEGBr4 Macroinitiators mPEG-Br linear macroinitiator was synthesized according to the literature procedure.[15] For the preparation of mPEG-Br2 and mPEG-Br4 macroinitiators, firstly, mPEG-OH2 and mPEG-OH4 were synthesized according to the literature procedure and characterized by 1H NMR.[15] In a typical procedure, mPEG-OH2 (1.25 g, 0.59 mmol; 1 equiv) was dissolved, under nitrogen atmosphere, in 15 mL of dry CH2Cl2 into a 100 mL two neck round-bottom flask equipped with condenser, dropping funnel, and magnetic stirrer. Then 0.22 g of DMAP (1.77 mmol) and 0.12 mL of TEA (0.88 mmol) were added and the reactor was thermostated at 0 8C. After cooling, 0.36 mL of BMPB (2.95 mmol; 5 equiv) in 5 mL of dry CH2Cl2 were added dropwise during 1 h. Subsequently, the temperature was allowed to raise room temperature and the reaction was continued under stirring for further 24 h. The solution was filtered and the product was precipitated in cold diethyl ether, filtered, washed with cold ethanol, and dried in vacuum. mPEG-Br2(Yield: 66%). 1H NMR(400 MHz, CDCl3): d 1.91 (s, CO(CH3)2Br); 3.70–3.91 (bs, –CH2–, m-PEG); mPEG-Br4 (Yield: 63%). 1H NMR(400 MHz, CDCl3): d 1.91 (s, CO(CH3)2Br); 3.70–3.91 (bs, –CH2–, m-PEG).

2.2.2. Synthesis of mPEG-(PMMA-ran-PAAEMA) Linear, mPEG-(PMMA-ran-PAAEMA)2, and mPEG-(PMMA-ranPAAEMA)4 Copolymers by ATRP mPEG-(PMMA-ran-PAAEMA) linear copolymers were synthesized in toluene at 90 8C. The reaction was carried out in a 100 mL glass flask charged, under nitrogen atmosphere, with 0.1 g of mPEG-Br linear macroinitiator in 15 mL of dry toluene. After the dissolution of the macroinitiator, 0.013 g of CuBr, 0.03 g of bpy, 5 mL of MMA, and 1.0 or 4.0 mL of AAEMA were added (0.5, 1.0, 2.5, or 4.0 mL were used in the case of DIPAEMA). The mixture was thermostated at 90 8C and magnetically stirred. The reaction was stopped with n-hexane after 18 h. The copolymer was recovered, dissolved in the minimum amount of chloroform, and passed over a column of activated Al2O3 to remove the catalyst. The solution was dried in vacuum, the polymer was washed with cold methanol, and then dried. mPEG-(PMMA-ran-PAAEMA)2 and mPEG-(PMMA-ran-PAAEMA)4 copolymers were synthesized using a molar ratio mPEG-Br2/CuBr/ bpy ¼ 1/4/8 and mPEG-Br4/CuBr/bpy ¼ 1/8/16, respectively. 1 H NMR (400 MHz, CDCl3): d 0.83-1.10(CH3 main chain), 1.79– 1.87 (CH2 main chain), 2.27 (–N(CH3)2), 2.56 (–O–CH2–CH2– N(CH3)2), 3.57 (–OCH3), 3.61(–OCH2CH2–), 4.06 (–O–CH2–CH2– N(CH3)2). 13C–NMR (400 MHz, CDCl3): d 16.6–18.9 (CH3 main chain), 44.9 (quaternary carbon in the main chain), 46.0 (–N(CH3)2), 52.0

ð1Þ

where DPmPEG ¼ MnðmPEGÞ =44; DPMMA ¼ DPmPEG ð2IMMA =I mPEG Þ; DPAAEMA ¼ DPmPEG ð2I AAEMA =nI mPEG Þ 44 is the molecular weight of monomeric units of mPEG; (ImPEG)/2 is half the integration of the signal relative to mPEG units: –OCH2CH2–; IMMA is the integration of the signal relative to MMA units: –OCH3; IAAEMA is the integration of methyl group of the signal relative to AAEMA units: –N(CH3)2, –N(CH2CH3)2, –N[CH(CH3)2]2 and –NHC(CH3)3, respectively; n indicates the number of methyl carbons of AAEMAs (2 for DMAEMA and DEAEMA, 3 for TBAEMA and 4 for DIPAEMA, respectively). Analogously, the monomers content in the copolymers were calculated using the following equations: X mPEG ffi I mPEG 2 I I mPEG = þI MMA þ AAEMA =n ; 2

X MMA ffi I

I MMA þ I AAEMA n ; =2 þI MMA

mPEG

X AAEMA ffi I AAEMA n I I mPEG = þI MMA þ AAEMA =n 2

ð2Þ

ð3Þ

ð4Þ

NMR spectra, GPC chromatogram and further information about formulas are provided in the Supporting Information (SI). The polydispersity index (PDI ¼ Mw/Mn) was evaluated by GPC.

2.3. NMR Analysis Spectra were recorded on a Bruker Avance 400 MHz spectrometer at 25 8C with D1 ¼ 5 s. The samples were prepared by introducing 20 mg of sample in 0.5 mL of CDCl3 into a tube (0.5 mm outer diameter). TMS was used as internal reference.

2.4. GPC Measurements The molecular weights (Mn) and the polydispersity index (PDI ¼ Mw/Mn) of polymer samples were measured by GPC at 30 8C, using THF as solvent, flow rate of eluant 1.0 mL min1, and narrow polystyrene standards as reference. The measurements were performed on a Waters 1525 binary system equipped with a Waters 2414 RI detector using four Styragel columns


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Mn ¼ DPmPEG ðMMEO Þ þ DPMMA ðMMMMA Þ þ DPAAEMA ðMMAAEMA Þ



(range 1 000–1 000 000 Å ). Every value was the average of two independent measurements.

2.5. Preparation of Plaques by Casting Plaques of 400 mm of thickness were prepared by dissolving 200 mg of polymer in 50 mL of CHCl3 at 25 8C. The solution was cast in a teflon petri dish (diameter 6 cm) and the solvent evaporated at room temperature. The plaque was removed from the petri dish and stored in a vacuum oven at 30 8C for 3 d. The resulting plaque is insoluble in water and does not leach bactericidal material.

2.6. Determination of Sorbed Benzoate[15] In a typical procedure the polymeric plaque (area 1 cm2; thickness 400 mm) was placed in 50 mL of an aqueous solution of benzoate sodium salt (3 ppm) under gentle stirring until saturation. Aliquots of benzoate solution were taken after 15 min, 30 min, 1 h, 2 h, 5 h, 24 h and then every 24 h until saturation. For each aliquot, the concentration was evaluated by measuring the absorbance at 224 nm using a value of 73 mM1 cm1 as the extinction coefficient. The amount of sorbed benzoate was evaluated by the difference with the starting concentration.

2.7. Determination of Surface Accessible Protonated Amines[2] The density of quaternary ammonium groups (QA, due to amine protonation) on plaque surfaces was measured evaluating the amount of fluorescein bound per unit area as reported in the literature. In a typical procedure the polymeric film (area 1 cm2; thickness 400 mm) was placed in 10 mL of an aqueous solution of fluorescein sodium salt (1 wt.-%) for 30 min under gentle stirring. The film was recovered and extensively rinsed with distilled water. Then the sample was placed in 3 mL of a solution of cetyltrimethylammonium chloride (0.1%) for 30 min until the film was colorless. To the corresponding solution were added 0.3 mL of a phosphate buffer solution 100 mM (pH 8.0) and the amount of the desorbed dye was evaluated by measuring the absorbance at 501 nm using a value of 77 mM1 cm1 as the extinction coefficient. The QA units on the film surfaces were evaluated considering the fluorescein concentration and a 1:1 ratio between fluorescein and QA.

2.8. UV–Vis Measurements To evaluate the amount of sorbed benzoate and fluorescein, UV–Vis measurements of the aqueous solutions were recorded on a Perkin Elmer Lambda EZ201 instrument, using PESSW 1.2 Revision E software, in the range 800–200 nm with a 1 cm quartz cells.

Luria-Bertani (LB) medium (10 g L1 trypton, 5 g L1 yeast extract, 10 g L1 NaCl) and grown aerobically for 12 h at 37 8C with constant shaking at 250 rpm. Subsequently, bacteria were collected by centrifugation for 10 min at 3 500 g, re-suspended at concentration of 0.01 OD600 in distilled, sterile water. To test for any material bactericidal power, the bacterial suspension (300 mL) was incubated in the presence of 1 cm2 of each polymeric plaque, (cut into four equal parts) at temperature of 37 8C with constant shaking at 250 rpm. A control with bacteria without polymer was also carried out. For cell survival determination at different time, 50 mL of suspension was spread on LB agar dishes (15 g L1 agar), incubated for 24 h and colony forming units (CFUs) calculated. All data were analyzed by statistical tests using Sigma plot 12.0 software. Statistical significance was tested by ANOVA and t-test. P-values under 0.05 were considered statistically significant. To evaluate possible polymers diffusion, experiments with conditioned supernatant were carried out. Plaques were incubated in distilled water with E. coli (0.01 OD600 mL1) for 24 h. Successively, residual bacteria were removed from water by centrifuging at 3 000 g for 5 min at 4 8C. To verify the presence of soluble molecules released from previous incubation, conditioned supernatant was inoculate with E. coli (0.01 OD600) from a fresh actively growing culture in absence of plaque and incubated for 24 h at 37 8C. Antimicrobial effect was evaluated by CFUs determination as reported above.

2.10. Hemolysis Test Toxicity to human red blood cells (RBCs) was estimated by a hemoglobin release assay and by the determination of amount of intact cells. RBCs were obtained from fresh human blood (1.2 mg mL1 of EDTA) by phosphate-buffered saline (PBS) (150 mM NaCl, 10 mM phoshate, pH 7.4) extraction. Briefly, human blood was diluted in PBS (1:10 v/v) and rinsed three times by centrifugation (10 min at 1 500 g). The collected cell suspension (1:10 v/v in PBS) was further diluted at 3% in PBS as RBCs stock suspension (S.s). For hemolysis test, each plaque was incubated with 50 mL of RBCs S.s diluted in 450 mL of PBS (0.3% RBCs s.s) and kept at 37 8C under constant shaking for different time.[22] To quantify the hemoglobin released, the samples were centrifuged for 5 min at 1500 g and the absorbance of the supernatant fluids was measured at 415 nm. To assess the plaque effects, the values were compared with those of 0.3% RBCs S.s. control sample, completely hemolysate by diluting RBCs in water bidistillated (1:10 v/v) and incubating at 37 8C for 30 min. Another sample of 0.3% RBCs S.s, incubated without plaque, was used as a blank to evaluate the lysis non-induced by polymers. To evaluate the amount of intact cells, the absorbance at 650 nm was measured using PBS buffer as a blank. To correct for cell lysis in absence of polymer plaques, a sample of 0.3% of RBC was also incubated in same condition used to test polymers but without the presence of the plaques.

2.11. Theoretical Modeling 2.9. Microbiological Assays The antimicrobial effects of the different polymers were evaluated as previously described.[15] Briefly, E. coli was pre-inoculated in

The free-energy change associated with the transfer of a proton from a protonated AAEMA group and a free one with different Nalkyl substituents, as well as the formation of a strong hydrogen


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bond between protonated and neutral AAEMA pendants (Scheme 1) bearing the same alkyl groups, are estimated using ab initio calculations at the MP2/6-311þþG(d,p)//MP2/6-31þG(d) level with the self-consistent reaction field/polarizable continuum model approach in Gaussian 09 and harmonic corrections estimated at the HF/6-31þG(d) level. This is a slight modification of what previously done to estimate similar quantities.[23] To limit costs and the bias due to the implicit solvent, we used in our model protonated and un-protonated mono-methyl di-alkyl amine, each alkyl group defining a specific AAEMA pendant, and two or four water molecules. The latter partially accounts for possible hydrogen bonds between the solutes and the aqueous solvent. We used four water molecules in the case of the secondary amine bearing the tert-butyl substituent, which can both accept and donate hydrogen bonds. The energy data obtained allow one to estimate the dimerization Gibbs energy change using DGdim ¼ Gðprotonated AAEMA dimerÞ þ Gðwater dimerÞ Gðprotonated AAEMA waterÞ GðAAEMA waterÞ: Instead, the change in free energy due to proton transfer between AAEMA pendants baring different substituents is given as DGPT ¼ GðprotonatedAAEMA0 waterÞ þ GðAAEMA00 waterÞ GðAAEMA0 waterÞ þ GðprotonatedAAEMA00 waterÞ In the previous equation, AAEMA0 and AAEMA00 represent pendants bearing different substituents, and DGPT should be correlated to the difference in pKb between the various AAEMA pendants.

AAEMA) were synthesized, casted into plaques, and the latter characterized and studied with respect to their antimicrobial activity. (Scheme 2). As mentioned, the mPEG would help producing the hydrophilic environment needed for the formation of the antimicrobial ammonium groups, while MMA, together with the high molecular weight, made copolymers nonsoluble in water and possibly improved the interaction between the polymeric surface and the hydrophobic part of the bacterial membrane. MMA was also chosen bearing in mind that water-soluble copolymers containing methacrylates showed the higher selectively for bacteria versus human cells with respect to copolymers with longer alkyl chains, e.g., butylmethacrylates.[17] The AAEMA monomers used here had the following Nalkyl substituents: –N(R1)(R2) with R1 ¼ R2 ¼–CH3 (Me), –CH2CH3 (Et), –CH(CH3)2 (i-Pr), and R1 ¼ H, R2 ¼–C(CH3)3 (tBu) (Scheme 2); hence, the amino groups were characterized by increasingly more hydrophobic substituents. All the copolymers were obtained by Atom Transfer Radical Polymerization (ATRP) of MMA and AAEMA using mPEG macroinitiators with different degree of branching at one chain-end. Two series of copolymers, having different amount of AAEMA in mol (40 and 15%), were synthesized and characterized by NMR (1H, 13C, and HSQC NMR, see SI).

3.2. The Hydrophobic/Cationic Charge Balance Modulated by the ‘‘Same Centered’’ Approach 3.2.1. Copolymers Containing 40% in mol of the AAEMA Monomer

3. Results and Discussion 3.1. Rational Design and Synthesis Branched A(BC)n copolymers (n ¼ 1, 2 or 4; ‘‘A’’ ¼ mPEG; ‘‘BC’’ ¼ random copolymeric chains based on MMA and

The microstructural characterization of copolymers containing 40% of AAEMA is reported in Table 1. The hydrophobic contribution of MMA was kept constant as

Scheme 2. Schematic representation and composition of the A(BC)n (n ¼ 1, 2, 4) copolymers.


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Table 1. Mn, chemical composition and polydispersity (Mw/Mn) of A(BC)n copolymers with 40% of AAEMA content.

Architecture A(BC) A(BC)2 A(BC)4 A(BC) A(BC)2 A(BC)4 A(BC) A(BC)2 A(BC)4 A(BC) A(BC)2 A(BC)4

AAEMA

Mn(NMR)a) [KDa] 76 96 93 70 87 153 81 84 105 93 110 130

XmPEGb)

[%]

XMMAb)

7.0 6.0 6.0 9.0 7.0 4.0 8.0 8.0 6.0 7.0 6.0 5.0

51 51 53 49 53 55 53 52 54 47 48 53

[%]

XAAEMAb)

[%]

42 43 41 42 40 41 39 40 40 46 46 42

Mw/Mnc) 1.5 1.5 1.8 1.5 1.8 1.8 1.4 1.4 1.4 1.5 1.5 1.8

a)

Evaluated from Equation (1); b)evaluated from Equation (2); c)evaluated from GPC. The molecular weight and the molar fraction of mPEG, MMA and AAEMA were calculated by the signal intensities I of full-assigned 13C NMR spectra (see Experimental Section). The GPC analysis of the products showed monomodal curves and molecular weights higher than that of the corresponding starting macroinitiators, confirming the presence of copolymers with a unique structure (see SI).

its molar fraction is unchanged; the hydrophobicity was instead tuned changing the N-substituents of the AAEMAs by the ‘‘same centered’’ approach. As it represents an important parameter, the total charge of plaques prepared from each copolymer by casting technique was evaluated by measuring the sorption of benzoate anion at saturation (Figure 1a). Apart from the copolymers containing i-Pr groups, data showed that the charge strongly depended on the architecture: the A(BC) plaques were the least charged, followed by the A(BC)4 ones. The A(BC)2 architectures showed to be the ones bearing the highest charge probably thanks to the higher propensity of forming strong hydrogen bonds between protonated and unprotonated amino groups, which increased the amount of charges both inside and on the plaque surface.[15] The finding that the sorbed benzoate increased over the time (Figure 1b) also suggests that the anion, probably, sorbes firstly and quickly on the surface, and only successively migrates slowly inside the bulk. The difference in sorption rate and capacity between the different copolymers can be interpreted as a difference in the density of surface and bulk charges. The lower effectiveness in absorbing benzoate shown by copolymers with i-Pr as N-alkyl groups, and the fact that it was found roughly independent of the architecture, was probably due to a lower charge density compared to other copolymers. This feature might be due to a different surface morphology, as supported by the fact that the most of the benzoate was apparently sorbed in the bulk, where, even here, protonable groups seem to have a limited accessibility. Given the similar benzoate sorption for all materials but the one with the i-Pr substituents, the effect of the

architecture on the antimicrobial activity was evaluated by incubating plaques containing t-Bu as Nsubstituent with suspended E. coli in distilled water for 1.5, 5, and 24 h under continuous shaking (Figure 1c). Notice that this approach requires bacteria to come in contact with the material surface due to suspension shaking, and are not directly deposited on it. The antimicrobial activity was in agreement with the charge density measurements of Figure 1a: the A(BC)2 architecture showed the highest antimicrobial effectiveness among the three materials, killing 87 14% of bacteria already after 5 h of exposure. This has to be compared to 43 7 and 59 5% for the A(BC) and A(BC)4 structures, respectively. The previously highlighted correlation between high benzoate sorption and strong antibacterial activity as a function of the structure suggests to evaluate the influence of the different N-alkyl substituents using plaques made of A(BC)2-type polymers (Figure 2). For these, the antimicrobial effect of Me, Et, and t-Bu N-groups was already evident after 1.5 h. At this time, the polymer with Me groups showed the highest antimicrobial power killing 91 9% of bacteria compared to 71 15 and 73 12% of Et and t-Bu groups, respectively. As the antimicrobial activity proceeds over the time, the differences between Me, Et, and t-Bu decreased significantly (after 5 h Me, Et, and t-Bu killed 94 6, 87 11, and 81 16% of bacteria, respectively; at 24 h, killed bacteria were 99.7 0.7, 97 6, and 99 1%, respectively). Interestingly, the polymer with i-Pr showed a bactericidal power comparable to that of the A(B)2 plaque which lacks any positive charge density and was used here for comparison purpose.


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Figure 1. Sorption of benzoate at saturation for the different architectures containing 40% of AAEMA (a); sorption of benzoate over the time until saturation of A(BC)2 structures (data were normalized with respect to the thickness of films N) (b); antimicrobial activity of copolymers with 40% of t-Bu as alkyl substituent at different times (c). Percentage values are referred to the number of CFU of control (E. coli without polymer) for each time. Values are means SD (n 3). Within each time (1.5, 5, and 24 h), statistically significant differences are labeled with different letters (in the group at 5 h, A(BC) vs. A(BC)2 t(6) ¼ 7.618, P < 0.001, A(BC)2 vs. A(BC)4 t(6) ¼ 6.889, P < 0.001).

Since we found a correlation between antimicrobial power and the sorption of benzoate, we decided to better quantify the charges encountered by bacteria in contact with the plaque surfaces evaluating the amount of surface accessible protonated amines using the fluorescein sodium salt method.[2] Data reported in Table 2 for copolymers with AAEMA 40% show the presence of similar surface charge densities for R1 ¼ R2 ¼ Me; Et and R1 ¼ H, R2 ¼ t-Bu in accordance with their antimicrobial behavior at 24 h as reported in Figure 2. However, since the copolymer with Me groups showed a higher antibacterial activity at 1.5 h of exposure to E. coli, it is reasonable to hyphotize that this was due to an easier accessibility of surface charges by bacteria, probably because of the less steric encumbrance of the methyl groups with respect to Et and t-Bu. Instead, the amount of surface charges was an order of magnitude lower for R1 ¼ R2 ¼ i-Pr, indicating the presence of a limited


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amount of accessible charges. Notably, the charge density values obtained with the fluorescein method correlate very well with the height of the intermediate plateaux appearing in Figure 1b, thus strongly supporting the idea that such feature was connected to surface adsorption. While the antimicrobial data showed in Figure 2 were in accordance with both benzoate and fluorescein sorption measurements, it is worth noting that the latter do not seem to follow the order in the pKb of the different amino-pedant groups. According to pKb values,[24] reported to be in the order DMAE (Me) > DEAE (Et) DiPAE (i-Pr) > TBAE (t-Bu), the N-methyl groups should confer the lowest basicity to the amino group and consequently the lower charge density to the copolymer. As suggested previously, this may be a telltale sign for the formation of charged hydrogen bonds, capable of stabilizing the excess protons in a way that depends on the capability of the amino-pedant groups to



Figure 2. Effects of the alkyl substituents on antimicrobial activity of A(BC)2 copolymers (40% of AAEMA). A(B)2 represents a copolymer without AAEMA. Percentage values are referred to the number of CFU of control (E. coli without polymer) for each time. Values are means SD (n 5). Within each time, statistically significant differences are labeled with different letters (in the group at 1.5 h, Me vs. A(B)2, P < 0.001, Me vs. Et, P < 0.001, Me vs. i-Pr, P < 0.001, Me vs. t-Bu, P < 0.001; in the group at 5 h, Me vs. A(B)2, P < 0.05, Me vs. i-Pr, P < 0.05, in the group a 24 h, Me vs. A(B)2, P < 0.05, Me vs. Et, P < 0.05, Me vs. i-Pr, P < 0.05).

dimerize.[15] In this respect, Table 3 provides the theoretical results for the formation free energy of a charged hydrogen bond between protonated and free amino-pendants, while Figure 3 shows the structure of the dimers. As can be seen, there is a net stabilization upon the formation of dimers in the case of all substituents except for iPr. The additional stabilization should thus increase the positive surface charge due to protonation compared to the non-dimerizing case.[25] Obviously, the strong effect predicted for t-Bu may be somewhat mitigated by the electrostatic interaction: an increase in the number of excess proton in the material would decrease the average distance between vicinal protonated dimers, the net effect of which would be to raise the chemical potential of the excess protons in the material due to repulsion. The equilibrium would then be reached at a lower protonation fraction than any theoretical estimate based on

dissolved species containing only two amino groups. From Figure 3, it is also apparent that the positive value for DGdim in the i-Pr case descends from the steric encumbrance of the four iPr groups. In this case, the two nitrogen atoms are forbidden to come sufficiently close to properly share the excess proton, an issue that is clearly not present in the complex with water (Figure 3d) due to its smaller size. 3.2.2. Copolymers Containing 15% in mol of the AAEMA Monomer Copolymers with low amount of AAEMA (15%) were synthesized to widen our investigation. The aim was to evaluate the effect of the N-alkyl groups in polymeric matrices where amino and ammonium groups are sufficiently diluted to reduce the probability of forming strong

Table 2. Surface charges of A(BC)2 films with 40 and 15% of the AAEMA content.

AAEMA

XAAEMA [%]

Surface charges/cm2

A(BC)2

R1 ¼ R2 ¼ Me

A(BC)2

R1 ¼ R2 ¼ Et

A(BC)2

R1 ¼ R2 ¼ i-Pr

A(BC)2

R1 ¼ H; R2 ¼ t-Bu

43 15 40 15 40 13 46 14

5.6 1014 2.2 1013 7.8 1014 1.7 1014 2.0 1013 1.4 1014 5.4 1014 1.7 1014

Architecture


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Table 3. Formation free energy (kJ/mol) for a charged hydrogen bond between protonated and free amino pendants.

Substituent DGdim

Me

Et

i-Pr

t-Bu

13.8

7.95

>0

20.9

hydrogen bond. The microstructural characterization of copolymers is reported in Table 4. Experiments showed that a lower amount of benzoate is sorbed by these copolymers than in the 40% case as a consequence of the lower amount of AAEMA (Figure 4a). At 15%, the copolymers with R1 ¼ R2 ¼ Me sorbed less benzoate than the others, this time in accordance with the order of pKb values; also, the effect due the polymer architecture seems to be absent (Figure 4b). The amount of surface charges (Table 2) is also lower for copolymers with N-methyl substituents, in agreement with the total amount of charge evaluated via benzoate sorption.

Microbiological tests indicated the plaques containing –N(CH3)2Hþ to be the least active among the different ammonium groups (Figure 5) even after 24 h (83 19% of killed cells compared to a mean value of 99.8 0.2% of the other three groups); this confirms again the strong correlation between the surface charge density and the antimicrobial activity. Consequently, amino-groups with a lower pKb value (i.e., containing Et, i-Pr and t-Bu N-groups) were the most active, killing an average of 96 5% of the cells compared to 52 29% of Me after 5 h in presence of bacteria. 3.3. The Hydrophobic/Cationic Charge Balance Modulated by the ‘‘Segregated Approach’’ 3.3.1. Comparison of Data for Copolymers with 15% and 40% of AAEMA Data in Figure 6 and Table 2 allow highlighting the influence of charge density or hydrophobicity, as

Figure 3. Optimized structure for the protonated amino dimer bearing the ethyl (a), t-Bu (b), i-Pr (c) substituents, and for the complex between the protonated i-Pr amino species and a water molecule (d). Distances (in Å ) between the excess proton and the hydrogen bond accepting atoms; notice the longer distance for the species in panel c.


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Table 4. Mn, chemical composition, and polydispersity (Mw/Mn) of A(BC)n copolymers with 15% of AAEMA content.

Architecture A(BC) A(BC)2 A(BC)4 A(BC) A(BC)2 A(BC)4 A(BC) A(BC)2 A(BC)4 A(BC) A(BC)2 A(BC)4 a)

AAEMA

Mn(NMR)a) [KDa]

XmPEGb) [%]

XMMAb) [%]

XAAEMAb) [%]

Mw/Mnc)

38 69 73 42 35 39 32 36 42 42 49 52

12 7.0 6.0 11 13 12 13 11 12 11 10 9.0

71 78 83 76 72 74 76 76 76 75 76 77

17 15 11 13 15 13 11 13 12 14 14 14

1.4 1.4 n.d. 1.4 1.3 1.2 1.3 1.2 1.2 n.d. 1.3 1.4

Evaluated from Equation (1); b)evaluated from Equation (2); c)evaluated from GPC.

modulated by the relative amount of protonable groups or hydrophobic MMA monomer (i.e., using the ‘‘segregated approach’’). The copolymer with 40% of AAEMA (50% of MMA) containing N-Me groups appeared as the most effective at short contact time (i.e., 1.5 h), a trend confirmed even after 5 h. However, reducing the load of protonable N-Me groups to 15% (i.e., increasing MMA content to 80%), substantially reduced the bactericidal action at 1.5 h (Figure 6a). Copolymers with i-Pr, Et, and t-Bu groups showed an antimicrobial activity fairly independent of AAEMA (and MMA) load. Interestingly, the copolymers with 15% of i-Pr or t-Bu (80% of MMA) showed a significant increase in their

antimicrobial activity at 5 h (Figure 6b) with respect to the analogue samples with lower amount of MMA. This behavior was more evident for the copolymer containing i-Pr groups, which surprisingly showed a higher surface charge density when containing a lower amount of i-Pr substituted groups (Table 2). Such change in effectiveness was not related to the swelling of the plaques as they were always pre-incubated in water before the microbiological tests. More in general, data in Table 2 show that the surface charge density was significantly lower than the average only for copolymers with i-Pr groups at 40% and Me groups at 15%. Both of them had a comparable charge density (2.0 1013–2.2 1013 charges/cm2); not surprisingly, these

Figure 4. Sorption of benzoate at saturation of the different architectures (a). Antimicrobial activity of copolymer with 15% of t-Bu as alkyl substituent at different times (b). Percentage values are referred to the number of CFU of control (E. coli without polymer) at each time. Values are means SD (n 3). Within each time, statistically significant differences are labeled with different letters (in the group at 1.5 h, A(BC)2 vs. A(BC)4 t(6) ¼ 2.709, P ¼ 0.035; in the group at 5 h, A(BC) vs. A(BC)2 t(6) ¼ 2.520, P ¼ 0.045).


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Figure 5. Effects of the alkyl substituents on antimicrobial activity of copolymer A(BC)2 (15% of AAEMA). A(B)2 represents a copolymer without AAEMA. Percentage values are referred to the number of CFU of control (E. coli without polymer) for each time. Values are means SD (n 5). Within each time, statistically significant differences are labeled with different letters (in the group at 1.5 h, t-Bu vs. A(B)2, P < 0.001, t-Bu vs. Me, P < 0.001; in the group 5 h, t-Bu vs. A(B)2, P < 0.001, t-Bu vs. Me, P < 0.001, and in the group 24 h, t-Bu vs. A(B)2, P < 0.001, t-Bu vs. Me, P < 0.05).

Figure 6. Antimicrobial activity of A(BC)2 copolymers with different percentage of AAEMA. Letters indicate the significant differences. a) after 1.5 h of contact with bacteria. (P < 0.001, according to to Holm–Sidak method). Values are means SD (n 5). b) After 5 h of contact with bacteria. (P < 0.05 according to Dunn’s Method). Percentage values are referred to the number of CFU of control (E. coli without polymer) for each time. Values are means SD (n 5).


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copolymers also correspond to the samples with lower antimicrobial activity. The results at 1.5 and 5 h thus seem to indicate that the hydrofobicity introduced by the increasing amount of MMA or by the alkyl substituents had no influence on the antimicrobial efficiency but rather that there is a threshold of surface-charge density above which the polymers start showing an appreciable bactericidal activity (1.4 1014 charges/cm2). Our findings are in accordance with experimental results € gler et al.[3] and by Murata et al.[26] relatively to reported by Ku glass surfaces coated with quaternized poly(vinylpyridine) and inorganic surfaces coated with quaternized polyDMAEMA, respectively. Both the authors agree in indicating the surface charge density as a crucial parameter to obtain surfaces with high killing efficiency and, more importantly, indicate the charge density threshold of the accessible positive charges for an optimal bactericidal efficiency of € gler reported that 1014 charges/ the coating. Specifically, Ku 2 cm were needed for killing about 100% of E. coli in lowdivision conditions when deposited directly on the modified surface. In high-division conditions, instead, the density required was much lower and of the order of 1012 charges/ cm2. Notice that the lower threshold, in the latter case, is due to both division conditions and the direct contact between bacteria and surface. Similarly, Murata[26] found that the polymeric surface, immobilized on inorganic materials and having greater than 5 1015 charges/cm2, was able to kill at least a monolayer of E. coli before fouling can take place. The authors also speculated that there was a convergence of numbers between the charge density of the cell surface (that is, between 5 1014 and 5 1015 charges/cm2 depending on the growth stage of the cells) and the charge density of an effective killing surface. Retrospectively, the charge densities reported in Table 2 seem also to indicate that the positive DG value for the formation of strong hydrogen bond in presence of the i-Pr N-groups at 40% is alone inadequate to rationalize the

very low surface-charge density and, consequently, the almost total absence of antimicrobial activity of the corresponding copolymer. In fact, considering that the pKb value of –N(i-Pr)2 is intermediate between that of –N(Me)2 and –N(t-Bu)H, it should provide a sufficient charge density when XAAEMA ¼ 40% to confer an appreciable antibacterial activity to the copolymer at least after 5 h of exposure to bacteria, as it does when XAAEMA ¼ 15%. It thus seems likely that such copolymer may have a different surface morphology from the others, as suggested earlier discussing the benzoate sorption. This difference may lead to a reduced exposition or accessibility of the protonable groups and, consequently, to a scarce antibacterial activity. Oddly, such an effect was not evidenced for copolymer with –NH(t-Bu) that contains just one bulky alkyl group instead of two. It is interesting to notice that the effect of surface morphology on antimicrobial activity has already been evidenced in the literature for coated surfaces. It is reported, in fact, that the presence of quaternizing alkyl chains longer than hexyl-groups diminished the antimicrobial power of the tetra-alkyl ammonium groups because of the chains collapse and a consequent charge shielding effect.[2,4–14] 3.4. The Hemolytic Activity of Antibacterial A(BC)2 Copolymers As reported previously, an optimum balance between cationic charge and hydrophobicity is required for watersoluble synthetic antimicrobial polymers in order to show antimicrobial but non-hemolytic properties.[16–19] In fact, while a strongly charged polycation may be cytotoxic toward human cells and can induce agglutination of red blood cells,[27,28] such tendency could be controlled compensating the lowering of the total charge with an increase of polymer hydrophobicity. Doing so, however, it is not without consequence, as increasing the hydrophobicity can raise the hemolytic character of the polymer as well; so that the fine tuning of the activity relies on the possibility of

Table 5. Hemolysis induced by A(BC)2 copolymers with different amount of AAEMA. The values are expressed as percentage of total hemolysis induced in the erythrocytes of control and are means SD (n ¼ 3). Polymeric plaque exposure area: 1 cm2.

AAEMA

Exposure time [h] 80% MMA

R1 ¼ R2 ¼ Me R1 ¼ R2 ¼ Et R1 ¼ R2 ¼ i-Pr R1 ¼ H; R2 ¼ t-Bu

50% MMA

5h

24 h

5h

24 h

2.51 2.73 0.00 0.00 0.00 0.00 5.18 7.11

0.21 0.36 0.28 0.49 0.81 1.41 11.2 11.6

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2.81 4.50 1.18 2.04 0.06 0.10 0.00 0.00


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increasing the bactericidal power faster than the hemolytic one. As a matter of fact, the absence of a hemolytic activity is a strong requirement for antimicrobial materials that may be applied in the biomedical field, so that we were interested in evaluating how the polymer charge density and hydrophobic character may affect such property for our antimicrobial copolymers. Plaques based on the A(BC)2 architectures containing 15 and 40% of AAEMA (corresponding to copolymers with 80 and 50% of MMA, respectively) were tested for their capacity to lyse human erythrocytes as a function of time. As shown in the Table 5, the amount of MMA and the nature of the N-substituents do not seem to influence significantly the hemolytic action, which is almost completely absent across the board even after long exposure time. Data were confirmed also by intact cell detemination, measuring the OD at 650 nm (see SI).

4. Conclusion In this paper, A(BC)n copolymers (n ¼ 1, 2 or 4; ‘‘A’’ ¼ mPEG, ‘‘BC’’ ¼ PMMA-ran-PAAEMA), were synthesized and characterized to be intrinsically antimicrobial. The antimicrobial activity as well as the hemolyticity of plaques casted with the different copolymers were evaluated varying the architecture, the nature of the alkyl-amino groups of the AAEMA, and the hydrophobic/charge density balance (given by the ratio MMA/AAEMA). We found that the A(BC)2 copolymers had the highest charge when containing a large amount of protonable groups (40% AAEMA), as such architecture is the one that allows a higher amount of proton bridges. The same rationale can explain the high effectiveness, shown just after 1.5 h of exposure to bacteria suspentions, of the copolymer containing 40% of AAEMA. Subtler (or not so subtle, with i-Pr substituents) differences, such as the higher effectiveness in presence of DMAEMA (N-methyl substituents) with respect to other AAEMA, may be due to variations in surface morphology or charge groups exposure. The architecture loses, instead, its influence on antimicrobial activity when a lower amount of AAEMA (15%) is present because of a reduced probability of forming proton bridges, and the efficiency of copolymers with different AAEMA appeared more correlated with the monomer pKb. More in general, the antimicrobial activity was dependent on the amount of surface charge density rather than on the hydrophobic/charge density balance, with a minimal surface-charge density of 1.4 1014 charges/cm2 to show an appreciable antimicrobial efficiency. It is also worth noting that, in contrast with water-soluble antimicrobial polymers, increasing the amount of hydrophobic MMA or the hydrophobicity of N-alkyl groups did not induce any hemolytic activity.

Ackowledgements: L.I. and G.V. are grateful for MIUR-FARB 2011 di Salerno; M.M. is grateful for MIUR-FAR funding from Universita dell’Insubria. L.I. is grateful for 2011 funding from Universita funding under the 2011 MIUR-PRIN scheme.

Received: November 18, 2014; Revised: January 9, 2015; Published online:DOI: 10.1002/mabi.201400503 Keywords: ATRP; antimicrobial polymers; non-hemolytic materials; self-protonable materials; star-polymers

[1] J. Huang, R. R. Koespel, H. Murata, W. Wu, S. Beom Lee, T. Kowalewski, A. J. Russel, K. Matyjaszewski, Langmuir 2008, 24, 6785. [2] J. C. Tiller, C.-J. Liao, K. Lewis, A. M. Klibanov, Proc. Natl. Acad. Sci. USA 2001, 98, 5981. € gler, O. Bouloussa, F. Rondelez, Microbiology 2005, 151, [3] R. Ku 1341. [4] N. M. Milovic, J. Wang, K. Lewis, A. M. Klibanov, Biotechnol. Bioeng. 2005, 90, 715.


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In conclusion, our results highlight that the charge density is a key parameter to obtain efficient thermoplastic, antimicrobial copolymers, and, perhaps more interestingly, that both the amount of charges and the presence of hydrophobic groups did not confer hemolityc power to the samples presented in this study. This is at variance with what happens with soluble species. Given the results, the A(BC)2 structures containing DMAEMA at 40% seem the most efficient, probably due to the low steric incumbrance of the methyl groups and the consequent spatial accessibility of the cationic charges; this finding may make it a good candidate as non-water soluble, highly-efficient antimicrobial, and non-hemolytic material with versatile structural capability for practical biomedical applications. The absence of leaching would guarantee both a good antimicrobial activity and a continuous re-activation of the material over the time, features that disclose the enormous potentiality of such copolymers as thermoplastic materials for medical devices. Contributions: C.V. has contributed to the polymer synthesis and characterization, as well as to the preparation of the plaques; S.M. has carried out the microbiological essays and analyzed the results; L.I. has designed the polymer structures and compositions, contributed to the polymer synthesis and characterization, analyzed the results of the latter, and participated to the writing of the manuscript; G.V. designed and carried out the microbiological essay, analyzed their results and contributed to the writing of the manuscript; M.M. has run the electronic structure calculations, analyzed their results, participated to the analysis of the physico-chemical characterization, and contributed to the writing of the manuscript.



[5] S. B. Lee, R. R. Koepsel, S. W. Morley, K. Matyjaszewski, Y. Sun, A. J. Russel, Biomacromolecules 2004, 5, 877. [6] J. C. Tiller, S. B. Lee, K. Lewis, A. M. Klibanov, Biotechnol. Bioeng. 2002, 79, 465. [7] J. Lin, J. C. Tiller, S. B. Lee, K. Lewis, A. M. Klibanov, Biotechnol. Lett. 2002, 24, 801. [8] L. Cen, K. G. Neoh, E. T. Kang, Langmuir 2003, 19, 10295. [9] F. X. Hu, K. G. Neoh, L. Cen, E. T. Kang, Biotech. Bioeng. 2005, 89, 474. [10] J. Lin, S. Qui, K. Lewis, A. M. Klibanov, Biotechnol. Prog. 2002, 18, 1082. [11] J. Lin, S. Qiu, K. Lewis, A. M. Klibanov, Biotechnol. Bioeng. 2003, 83, 168. [12] J. Thome, A. Hollander, W. Jaeger, I. Trick, C. Oehr, Surf. Coat. Tech. 2003, 174–175, 584. [13] J. Lin, S. K. Murthy, B. D. Olsen, K. K. Gleason, A. M. Klibanov, Biotechnol. Lett. 2003, 25, 1661. [14] M. Ignatova, S. Voccia, B. Gilbert, N. Markova, P. S. Mercuri, M. Galleni, Langmuir 2004, 20, 10718. [15] G. Vigliotta, M. Mella, D. Rega, L. Izzo, Biomacromolecules 2012, 13, 833. [16] K. Kuroda, G. A. Caputo, W. F. DeGrado, Chem. Eur. J. 2009, 15, 1123.

[17] E. F. Palermo, K. Kuroda, Biomacromolecules 2009, 10, 1416. [18] E. F. Palermo, I. Sovadinova, K. Kuroda, Biomacromolecules 2009, 10, 3098. [19] B. P. Movery, S. E. Lee, D. A. Kissounko, R. F. Epand, R. M. Epand, B. Weisblum, S. S. Stahl, S. H. Gellman, J. Am. Chem. Soc. 2007, 129, 15474. [20] G. J. Gabriel, J. A. Maegerlin, C. F. Nelson, J. M. Dabkowski, T. € sslein, G. N. Tew, Chem. Eur. J. 2009, 15, Eren, K. Nu 433. [21] V. Sambhy, B. R. Peterson, A. Sen, Angew. Chem. Int. Ed. 2008, 47, 1250. [22] I. Sovadinova, E. F. Palermo, R. Huang, L. M. Thoma, K. Kuroda, Biomacromolecules 2011, 12, 260. [23] A. Ponti, M. Mella, J. Phys. Chem. A 2003, 107, 7589. [24] H.-L. Chen, K. C. Hou, React. Polym. Ion Exch. Sorb. 1987, 5, 5. [25] M. Mella, L. Mollica, L. Izzo, J. Polym. Sci. Part B: Polym. Phys., 2015, http://dx.doi.org/10.1002/polb.23680. [26] H. Murata, R. R. Koespel, K. Matyjaszewski, A. J. Russel, Biomaterials 2007, 28, 4870. [27] D. Fischer, X. Y. Li, B. Ahlemeyer, J. Krieglstein, T. Kissel, Biomaterials 2003, 24, 1121. [28] E. Moreau, M. Domurado, P. Chapon, M. Vert, D. Domurado, J. Drug Target. 2002, 10, 161.


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