Branched High Molecular Weight Glycopolypeptide With Broad

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Branched High Molecular Weight Glyco-polypeptide With Broad-spectrum Antimicrobial Activity for the Treatment of Biofilm Related Infections Nicolò Mauro, Domenico Schillaci, Paola Varvarà, Maria Grazia Cusimano, Daniela Maria Geraci, Mario Giuffrè, Gennara Cavallaro, Carmelo Massimo Maida, and Gaetano Giammona ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16573 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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ACS Applied Materials & Interfaces

Branched High Molecular Weight Glyco-polypeptide With Broad-spectrum Antimicrobial Activity for the Treatment of Biofilm Related Infections Nicolò Mauro,a* Domenico Schillaci,b Paola Varvarà,a Maria Grazia Cusimano,b Daniela Maria Geraci,c Mario Giuffrè,c Gennara Cavallaro,a Carmelo Massimo Maida,c Gaetano Giammonaad a

Laboratory of Biocompatible Polymers, Department of “Scienze e Tecnologie Biologiche,

Chimiche e Farmaceutiche” (STEBICEF), University of Palermo, Via Archirafi, 32 90123 Palermo, Italy. b

Department of “Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche” (STEBICEF),

University of Palermo, Via Archirafi, 32 90123 Palermo, Italy. c

Department of “Scienze per la Promozione della Salute e Materno Infantile - G. D'Alessandro”

University of Palermo, Via del Vespro 133, 90127 Palermo, Italy. d

Mediterranean Center for Human Advanced Biotechnologies (Med-Chab), Viale delle Scienze

Ed.18, 90128 Palermo, Italy. *Corresponding author: [email protected] Fax: +39 09123891928; Tel: +39 09123891928 KEYWORDS Antimicrobial polymers, synthetic polypeptides, colistin, vancomycin, Pseudomonas aeruginosa, Staphylococcus aureus, Biofilms ABSTRACT There are few therapeutic options to simultaneously tackle Staphylococcus aureus and Pseudomonas aeruginosa, two of the most relevant nosocomial and antibiotic-resistant pathogens responsible for implant, catheters and wound severe infections. The design and synthesis of 1 ACS Paragon Plus Environment

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polymers with inherent antimicrobial activity have gained increasing attention as a safe strategy to treat multidrug-resistant microbes. Here, we tested the activity of a new polymeric derivative with glyco-polypeptide architecture (PAA-VC) bearing L-arginine, vancomycin and colistin as side chains acting against multiple targets, which give rise to a broad spectrum antimicrobial activity favorably combining specific and nonspecific perturbation of the bacterial membrane. PAA-VC has been tested against planktonic and established biofilms of reference strains S.aureus ATCC 25923 and P.aeruginosa ATCC 15442 and susceptible or antibiotic resistant clinical isolates of the above mentioned microorganisms. MIC values observed for the conjugate (48 – 190 and 95 – 190 nM for P. aeruginosa and S. aureus strains respectively) showed higher efficacy if compared with the free vancomycin (MICs within 1.07 – 4.28 µM) and colistin (MICs within 0.63 – 1.33 µM). Additionally, being highly biocompatible (IC50 > 1000, 430 and 250 µg mL-1 for PAA-VC, vancomycin and colistin respectively) high-dosage can be adopted for the eradication of infections in patients. This positively influences the antibiofilm activity of the conjugate leading to a quasitotal eradication of established clinically relevant biofilms (inhibition > 90% at 500 µg mL-1). We believe that the in vitro presented data, especially the activity against established biofilms of two relevant pathogens, the high biocompatibility and the good mucoadhesion properties, would allow the use of PAA-VC as promising candidate to successfully address emerging infections. INTRODUCTION Nowadays, the antibiotic-resistance of common pathogens has weakened our capacity to control microorganisms and therefore treatment and prevention of infections. For instance, severe infections derived from using implants and medical devices (e. g., prosthesis and catheters) are often due to antibiotic resistant bacteria.1,2 Both Gram positive and Gram negative bacteria may be responsible of severe infections and therapeutic strategies are limited. Besides, chronic pulmonary, dental3 and vaginal diseases can be associated to the spread of antibiotic resistant colonies which persist in the host organism. P. aeruginosa is a common cause of health care–associated infections 2 ACS Paragon Plus Environment

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(HAIs), including pneumonia and bloodstream, urinary tract, and surgical-site infections.4 More than 6,000 (13%) of the 51,000 HAIs P. aeruginosa infections that occur in the U.S. each year are multiple drug resistant (MDR).5 Moreover, Methicillin-Resistant S. aureus (MRSA) has spread worldwide. It is one of the major pathogen liable for HAI and it is resistant to penicillin-like betalactam antibiotics. There are few therapeutic options to simultaneously tackle these two relevant pathogens to human health and no efficient treatment has been identified so far. Finally, it is a matter of fact that in such infections, for example on wound healing, the microbial growth as a complex sessile community (biofilm) is prevalent, implying much higher maximum concentrations of antibiotics and prolonged periods of action for eradicating bacteria in biofilms compared to planktonic cells. This is usually not feasible in vivo owing to toxicity issues of common antibiotics.6,7 Severe infections in critical patiens (e.g. patients in intensive care units or eith extensive skin lesions) often require a sudden empirical antimicrobial treatment in the absence of a diagnostic address towards etiology (Gram+ or Gram-). The therapeutic choice is always difficult and complicated by patient conditions and tolerability or drug side effects, duration of treatment and selection of multidrug resistant bacteria, according to most recent protocols of antimicrobial stewardship.8 The design and synthesis of polymers with antimicrobial activity have gained increasing attention as a safe strategy to treat multidrug-resistant microbes, since they exhibit long-term activity.9–12 Polymers with intrinsic antimicrobial activity are usually based on polycations carrying quaternary ammonium, quaternary phosphonium, guanidine or tertiary sulfonium side groups, which are able to kill microbes by means of electrostatic interactions with negative charges exposed on the bacterial membrane. Despite the good activity associated with polycations, they are strongly basic and sometimes toxic, which limit their use to short treatments and low dosage.13,14 Among these, arginine-rich peptides (ARPs) are a particular class of antimicrobial polymers that form transmembrane pores through the interaction of their guanidine side functions with bacterial lipidic 3 ACS Paragon Plus Environment


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core.15–18 They have attracted great attention as they display broad and potent antimicrobial activity. Unfortunately, the multistep synthetic pathway employed for the synthesis of such peptides leads to high production costs, which is also associated with poor bioavailability in vivo because of their proteolytic degradation under physiological conditions.19 Someone might argue that the conjugation of arginine with biocompatible high molecular weight polymers is probably useful to impart ARPs properties while circumventing hydrolytic inactivation in vivo as well as toxicity phenomena. We have previously developed a cost-effective synthetic branched copolymers with polypeptidic structure, named poly(argilylaspartamide)aspartic acid (PAA), consisting of a polyaspartic acid backbone statistically derivatized with arginine via amidic bond as side chain mimic of ARPs.20 Arginine residues endow PAA with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of the bacterial membrane.21 However, this copolymer is intrinsically amphoteric and prevailingly anionic under physiological conditions, as it exhibits one carboxylic function (pKa = 6.1, 4.4, 3.3, arginine, β and α aspartic acid respectively) per repeating unit and about 0.7 guanidine groups (pKa ~ 11.7 ),20 and it is not able to interact with the negatively charged bacterial membrane implying a negligible antimicrobial effect. While, the amphoteric nature of PAA is concurrently responsible for the emphasized biocompatibility showed by these polypeptides (IC50 > 10 mg mL-1 after 7 days incubation).17,20,22 This scenario prompted us to think about a high molecular weight PAA-based conjugate, named PAA-VC, involving the combination of a glyco-peptide and a peptide side chain with antimicrobial activity (i.e., colistin and vancomycin) acting against multiple targets, which give rise to broad spectrum antimicrobial activity toward established biofilms. In this study, we assessed the enhanced activity of PAA-VC against established biofilms of S. aureus and P. aeruginosa, the most critical and priority group of pathogens classified in the WHO list,23 and compared its anti-biofilm activity with the pristine vancomycin and colistin. In February 2017, WHO published its first ever list

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antibiotic-resistant “priority pathogens”, a catalogue of 12 families of bacteria that pose the greatest threat human health. MATERIALS AND METHODS D,L-aspartic acid (98%), phosphoric acid (85%), N,N-dimethylformamide (DMF), vancomicin hydrochloride (USP), colistin sulfate (> 15 kU mg-1), colistin methanesulfonate (15.5 kU mg-1), Larginine (99%), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 98%), Nhydroxysuccinimide (NHS, 98%), acetonitrile (ChromasolvTM), sodium sulfate (99%), MuellerHinton broth (MHB), cation adjusted Mueller-Hinton broth (MHB2), Tryptic Soy Broth (TSB) and crystal-violet were purchased from Sigma Aldrich and used as received. 1

H NMR spectra were recorded using a Bruker Avance II 300 spectrometer operating at 300.12

MHz. Size exclusion chromatography (SEC) traces were obtained using Tosho Bioscience TSK-Gel G4000 PWXL and G3000 PWXL columns connected to an Agilent 1260 Infinity Multi-Detector GPC/SEC system. Right angle/low angle laser and refractive index detectors were employed. The mobile phase was a 0.1 M Tris buffer pH 8.10 ± 0.05 with 0.2 M sodium chloride. The flow rate was 0.6 mL min-1 and sample concentration 15 mg mL-1. The absolute molecular weight of the polymers were calculated using the Rayleigh’s scattering equation. S.aureus ATCC 25923 and P.aeruginosa ATCC 15442, reference strains in official tests for antibacterial evaluation in vitro (UNI EN European Standard), were purchased from PBI-VWR (Italy). Syto9 and propidium iodide were purchased from Molecular Probes Inc., Eugene, OR, USA and used as received. Synthesis of Poly(argilylaspartamide-co-aspartic) acid (PAA). PAA was synthetized as previously reported.20 Briefly, polysuccinimide (PSI) was obtained by polycondensation of D,Laspartic acid in phosphoric acid.24 A solution of PSI (200 mg, 2.062 mmol) in DMF (4 mL) was 5 ACS Paragon Plus Environment


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added dropwise to a solution of L-arginine (1.0000 g, 5.740 mmol) in ultrapure water (10 mL) at pH 10.50 ± 0.05. After 24 h at 22-28 °C, the product was dialyzed against water using a membrane with 50 kDa nominal molecular weight cutoff (NMWC) and then freeze-died. Yield 360 mg (89 %). 1

H NMR (D2O, 300 MHz): δH 1.62 (2Harg, br, CHCH2CH2), 1.85 (2Harg, m, CHCH2CH2), 2.66

(2Hβ

aspartic acid,

br, NHCOCH2CH), 2.72 (2Hamide, br, a and b CH2; 2Hα

(2Harg, t, CH2NHCN2H4), 3.73 (1Harg, t, CHCOO-), 4.44 (1Hβ

aspartic acid,

aspartic acid,

CH2COO-), 3.15

br, NHCOCH2CH), 4.62

(1Hα aspartic acid, br, CHCH2COOH and 1Haspartamide, α and β CH). Size Exclusion Chromatography: Mw = 62100, PD = 1.41. Synthesis of Poly(argilylaspartamide-co-aspartic) acid-colistin Derivative (PAA-C). PAA (100 mg) was dissolved in water (2 mL) and the pH was adjusted to 6.8 using 1M sodium hydroxide (37.5 µL). Then, EDC (3 mg, 0.015 mmol) and NHS (1.8 mg, 0.015 mmol) were added to this solution at once. Separately, colistin sulfate (25 mg, 0.020 mmol) was solubilized in PBS pH 6.8 (500 µL) and added to the reacting mixture dropwise. The reaction was kept at r.t. (min 16, max 20 °C) for 18 h. After purification through a membrane dialysis with 25 k NMWC the crude was freeze-dried, obtaining a yellowish powder. Yield 108 mg (87 %). 1

H NMR (D2O, 300.15 MHz): δH 0.82 (m, 18Hcolistin;), 0.92 (10Hcolistin, m, CH2CHCH2 and CH3),

1.19 (2Harg, br, CHCH2CH2 and 4Hcolistin CH3CHCH2) 1.58 (2Harg, m, CHCH2CH2; 1Hcolistin, CH3CH) 1.86 (11Hcolistin CH2CH2NH2, CH3CHCH2) 2.15 (8Hcolistin, m, CH3CHOH and NHCOCH2), 2.21-2.29 (10Hcolistin CH2CH2NH2), 2.70 (2Hbackbone, br, CH2COO- , NHCOCH2CH), 3.15 (3Harg, br, CH2NHCN2H4 and CHCOO-), 3.25 (4Hcolistin, NHCH2CH2CH), 4.15 (2Hcolistin, br, CH3CHOH), 4.21 (8Hcolistin, br, NHCOCHNH), 4.34 (2Hcolistin, br, OHCHCH), 4.49 (1Hbackbone, s, NHCOCH2CH), 4.64 (CHCH2COOH). Size Exclusion Chromatography: Mw = 65200, PD = 1.40.


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Synthesis of Poly(argilylaspartamide-co-aspartic) acid-vancomycin,colistin Derivative (PAAVC). EDC (14.4 mg, 0.075 mmol) and NHS (8.65 mg, 0.075 mmol) were added to a solution of PAA-C (80 mg) in PBS pH 6.8 (4 mL) under vigorous stirring. Separately, vancomycin hydrochloride (29.8 mg, 0.02 mmol) was solubilized in ultrapure water (500 µL) and added to the reaction under stirring. The reaction mixture was then maintained at r.t. (min 15, max 21 °C) with gentile stirring for 18 h. After this time, it was dialyzed against ultrapure water using a test tube with 50 kDa NMWC. The product was lyophilized and obtained as a whitish powder. Yield 81 mg (74 %). 1

H NMR (D2O/DMF 8:2, 300.15 MHz): δH 0.5-0.87 (m, 18Hcolistin; 6Hvancomycin, CH3 and

3Hvancomycin, CH3), 0.99 (6Hcolistin, m, CH3), 1.15 (2Harg, br, CHCH2CH2 and 4Hcolistin CH3CHCH2), 1.56 (2Harg, br, CHCH2CH2; 1Hcolistin, CH3CH) 1.83 (2Harg, m, CHCH2CH2; 11Hcolistin CH2CH2NH2, CH3CHCH2;

3Hvancomycin,

CH3CHCH2),

1.92-2.38

(21Hcolistin

CH2CH2NH2,

CH3CHCH2,

CH3CHOH and NHCOCH2), 2.69 (2Hbackbone, br, CH2COO- , NHCOCH2CH), 2.73 (3Hvancomycin, s, CH3-N),

3.15

(2Harg,

t,

CH2NHCN2H4),

3.25-3.62

(1Hvancomycin,

NHCHCO;

4Hcolistin,

NHCH2CH2CH), 3.70 (br, 1Harg, br, CHCOO-; 3Hvancomycin, OCHCH2 glycosyl moieties), 6.40 (4Haromatic

ring

, br., vancomycin), 6.92 (4Haromatic

ring

, br., vancomycin), 7.31 (5Haromatic

ring,

br.,

vancomycin). Size Exclusion Chromatography: Mw = 67100, PD = 1.34. Z-potential (ζ ζ) Measurements. ζ measures were performed using a Malvern NanoZS instrument. Samples were prepared in water at concentration of 0.1 mg mL-1. The electrophoretic mobility of the particles was evaluated for 2 min at 25°C, applying a constant electric field. Each sample was analyzed in triplicate, and the average Z–potential reported. The zeta-potential (mV) was calculated from the electrophoretic mobility using the Smoluchowsky relationship and assuming that K × a >> 1 (where K and a are the Debye-Hückel parameter and particle radius, respectively).25



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Evaluation of the Chemical Stability of PAA-VC in Physiological Conditions. The PAA-VC conjugate (5 mg) was dissolved in PBS pH 7.4 (5 mL) and kept at 37 °C under continuous stirring (100 rpm) in a Benchtop 808C Incubator Orbital Shaker model 420, for 7 days. At intervals (1, 3, 5, 7 days) 100 µL solution were withdrawn, freeze-dried, and then analyzed by HPLC after dissolving in the eluent.26 For the HPLC analysis the mobile phase was 30 mM Na2SO4/H2PO4- buffer solution pH 2.5: acetonitrile (80:20 % v/v) at the flow rate of 0.8 mL min-1. The mobile phase was freshly filtered through a 0.22 µm membrane filter before performing the analysis. The column was Luna® C18 5 µm pore size, equilibrated at 25 °C during the separation. The detection of species was performed by an online UV spectrometer within the range 190 – 400 nm, step 2 nm, and the injection volume was 50 µL (Agilent 1200 Infinity). Mucoadhesive properties of PAA-VC. A solution of PAA-VC in PBS pH 7.4 was added to a solution of mucin in the same buffer under vigorous stirring (mucin and PAA-VC final concentrations, 1 and 0.05 or 0.20 mg mL-1 respectively). The obtained colloidal dispersions were incubated at 37 °C and the interaction between mucin and the conjugate was determined at scheduled time intervals (0, 25, 50, 150 and 300 min), as a measure of the amount of cloudiness observed, by turbidimetric measurements using a Shimadzu-3600 UV Spectrophotometer: λ = 495 nm. A solution of mucin in PBS pH 7.4 (1 mg mL-1) was evaluated as a negative control. Results were obtained in triplicate and expressed in terms of transmittance percentage. Cytotoxicity Assay. Human bronchial epithelium (16 HBE) cells were purchased by Lifetechnologies (Thermo Fisher Scientific Inc.) and cultured in complete DMEM (DMEM supplemented with 10% v/v of FBS, 1% v/v of penicillin–streptomycin solution, 1% v/v of glutamine solution and 0.1% v/v amphotericin B solution.) following the supplier instructions. Cytocompatibility tests were carried out incubating cells with complete DMEM solutions of PAAVC. In particular, 1 x 104 cells were seeded into each well of 96-well plate and cultured for 24 h before testing. After 24 h, the medium was changed with complete DMEM solutions of copolymer 8 ACS Paragon Plus Environment

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at concentration ranging from 0.1 to 10.0 mg mL-1 and the cell viability was evaluated after 24 and 48 h of incubation, by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS) (Promega). Chronic contact cytocompatibility tests were performed incubating cells with the polymeric solutions for 2 and 7 days, with the preventative measure of refreshing the polymeric solutions after 3 days of incubation. For all the experiments, positive control was made by culturing 1 x 104 16 HBE in complete DMEM without any other supplement. Minimum Inhibitory Concentrations (MICs) Determination. MICs of the PAA-VC conjugate and reference antibiotics vancomycin chloridrate and colistin sulphate toward free living forms (planktonic) of two clinical strains of S. aureus (one methicillin sensitive - MSSA and one methicillin resistant - MRSA), two clinical strains of P. aeruginosa (one cephalosporin sensitive and one metallo-beta-lactamse producing - M βL) and reference strains of S. aureus (ATCC 25923) and P. aeruginosa (ATCC 15442) were determined by using a reference method for dilution antimicrobial susceptibility test for bacteria that grow aerobically (CLSI)27 with some modifications. Briefly, 0.1 mL of a sterile stock solution (400 µg mL-1) of antimicrobials, that is either vancomycin hydrochloride, colistin sulfate or PAA-VC, was added into a well of sterile 96wells plate and 1:2 dilution series with broth medium was performed. A growth control and a sterile well were included per each plate. The direct colony suspension method was performed for the preparation of the inoculum. Broth suspensions in Trypticasein Soy Broth (Laboratorios Conda S.A., Madrid, Spain) of isolated colonies, selected from a 18 to 24-hours agar plate, were adjusted to achieve a turbidity equivalent to a 0.5 McFarland standard. This results in a suspension containing approximately 1 to 2 x 108 colony-forming units (CFU) mL-1. A 1:20 dilution procedure was used to yield inoculum concentration of 5 x 106 CFU mL-1. When 0.01 mL of this suspension was inoculated into the broth, the final test concentration of bacteria was approximately 5 x 104 CFU mL-1 (0.01 mL of bacterial inoculum into 0.1 mL of antimicrobials). Colony counts on 9 ACS Paragon Plus Environment


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inoculum suspensions were performed to ensure that the final inoculum concentration obtained was 5 x 106 CFU mL-1. For this purpose, 0.01 mL aliquot from the growth control tube was diluted into 10 ml of saline solution (1:1000 dilution) and 0.1 mL aliquot was spread over the surface of a Muller-Hinton agar medium. After incubation, the presence of approximately 50 colonies indicated an inoculum density of 5 x 105 CFU mL-1. 96-wells plates were incubated at 35±2°C for 24 hours and MICs (the lowest concentration of antimicrobial agents that completely inhibits growth of the organism in the microdilution wells referred to control wells) values were recorded. Inhibition of Biofilm Formation, Crystal Violet Method. Bacterial strains were incubated in test tubes with TSB (5 mL) containing 2% w/v glucose at 37 °C for 24 h. After that, the bacterial suspensions were diluted to achieve a turbidity equivalent to a 0.5 McFarland standard. The diluted suspension (2.5 µl) was added to each well of a single cell culture polystyrene sterile, flat-bottom 96-well plate filled with TSB (100 µl) with 2% w/v glucose. Sub-MIC concentration values of all substances were directly added to reach final concentrations of 12.5; 6.25; 3.125 and 1.6 µg mL-1 for conjugate, 6.5; 3.125; 1.6 0.8 and 0.4 µg mL-1for colistin sulfate and 6.25; 3.125; 1.6, 0.8 and 0.4 µg mL-1 for vancomycin. Plates were incubated at 37°C for 24h. After biofilm growth, the content of each well was removed, wells were washed twice with sterile PBS 1X and stained with 150 µl of 0.1% w/v crystal violet solution for 30 min.28 at room temperature. Excess solution was removed and the plate was washed twice using tap water. A volume of 125µl of acetic acid of 33% v/v was added for 15 min to each stained well to solubilize the dye. Optical density (OD) was read with wavelength of 540 nm using a microplate reader (Labsystem Multiskan® MCC/340). To calculate the percentages of inhibition the following formula was used:

% of Inhibition =

OD − OD 100 OD

Anti-biofilm Activity, Crystal Violet Method. A suspension of bacteria (0.5 McFarland standard) was obtained using the procedure described above for the inhibition of biofilm formation test. 2.5 µl 10 ACS Paragon Plus Environment

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of suspension was added to each well of a 96-weel plate containing TSB (100 µl) with 2% w/v glucose. After the growth of a biofilm (24h old), the content of each well was removed, wells were washed up twice with sterile PBS and then filled with fresh TSB medium (200 µl). After that, different concentrations of PAA-VC or reference antibiotics were added starting from a concentration equal or greater than MIC obtained against planktonic form of tested strains using TSB as medium. The microtiter plate was sealed and incubated at 37°C for further 24 h. The content of each well was removed, wells were washed up twice with sterile PBS (100 µl to each well) and the 96-weel plate was placed at 60 °C for 1 h before staining with a 0.1% w/v crystal violet solution. After 30 min, plates were washed with tap water to remove any excess stain. Biofilm formation was determined by solubilizing crystal violet staining in 33% v/v acetic acid (125 µl) for 15 min and measuring the absorbance at 540 nm using a microplate reader (Labsystem Multiskan®MCC/340). To calculate the percentages of inhibition the formula above reported was used. Biofilms Susceptibility Testing, Static Chamber System. Cultures of S. aureus or P.aeruginosa were grown in TSB containing glucose at 2 % w/v overnight at 37°C in a shaking bath and then diluted 1:200 to a suspension with optical density (OD) of about 0.040 at 570 nm corresponding to ~106 CFU/mL. Then cover glass cell chambers (2 wells) were inoculated with 1.5 mL per well of above suspensions and incubated at 37°C for 24 h. After incubation, the medium was removed, chambers were washed up gently four times with PBS, and fresh medium containing 50 µg mL-1 of tested PAA-VC conjugate or free vancomycin or free colistin sulphate were added and incubated at 37 °C for further 24h. At the end of incubation time the medium was removed, chambers were washed up and then stained by 1 µM SYTO9 (green fluorescent stain for living cells) and 1 µM propidium iodide (PI) (red fluorescent stain for dead cells) for 30 min and observed under a confocal microscope (Olympus FV10i) equipped with a 60x objective. Z-stack images were



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collected and analyzed to evaluate cell viability at different depths. At least five images were taken at randomly chosen locations over the surface of the glass slide.29 RESULTS AND DISCUSSION Rationale. Polysuccinimide ring opening is normally performed by nucleophilic attack of amines, thiols or hydrazine in aprotic polar solvents such as DMF, at controlled temperature (22-28 °C) and without adding catalysts. In principle, high functional branched polypeptide with a polyaspartic acid backbone bearing L-arginine as side chain can be obtained, since under these conditions carboxyl and guanidine groups, being ionized, would not interfere in the ring-opening reaction directed by the α-amino function (Scheme 1). However, the way in which such polypeptides are obtained is not trivial, because α-amino acids are not soluble in aprotic polar solvents and have lower pKa values and higher steric hindrance than common primary amines (pKa = 9.4 vs pKa = 10.5), entailing a weak nucleophilicity.13,30 For these reasons, the synthetic pathway that leads to such polypeptides (PAA) consisting in statistically arranged α and β aspartic units copolymerized with α and β argilyl-aspartamide repeating units involve the hetero-phase reaction between L-arginine and polysuccinimide (PSI) in H2O/DMF 72:28 (Scheme 1), since this particular solvent mixture allows complete dissolution of the amino acid still maintaining a good solubility of PSI during the reaction. This reaction can be also kinetically controlled by tuning the pH of the reaction, yielding to PAAs with Mw ranging from 10 to 67 kDa for pH 8.5 and 10.5 , respectively.20 This polypeptide aroused our interest because it is a biocompatible inherently multifunctional arginine-rich polymer potentially capable of destabilizing the bacterial membrane.20 Arginine-rich polymers like PAAs are endowed with good antimicrobial activity, since the guanidine side groups destabilize bacterial membrane and wall provoking disruption of cells. However, it is well-known that the mechanism of the bactericidal action of polyelectrolytes involves strong destabilizing interactions with the cell wall or cytoplasmic membranes (net negative surface charge); hence it is generally accepted that only positively charged macromolecules may act as antimicrobial 12 ACS Paragon Plus Environment

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polymers.31 Under physiological conditions PAA is prevailingly anionic (ζ = -21.2 mV) and guanidine functions are counterbalanced by carboxylic groups of aspartic repeating units (Scheme 1, orange units) or closely related arginine pendants (Scheme 1, purple units). Thereby, owing to long-range electrostatic repulsion phenomena, the interaction between guanidine side functions and negatively charged bacterial cell membranes may not occur penalizing theirs ARPs-like biocidal properties (Supporting information, Table S2, Pag. S-4). On the other hand, appealing features of PAAs include the possibility to fine-tune the surface charge and biological properties by varying composition of side chains. Indeed, each repeating units contain a reactive carboxyl moiety amenable to further functionalization. This is particularly important since it is possible to simultaneously conjugate to the main backbone molecules acting as antimicrobials against different targets avoiding antibiotic-resistance mechanisms. Besides, the use of cationic side actives may lead to polyampholytes with cationic behavior that allows arginine to play a role as additional nonspecific destabilizing agent. Taking this in mind, we thought to tune the antimicrobial activity of the main polypeptide (PAA) by choosing a Gram-positive active glycosylated peptide and a Gram-negative active peptide, namely vancomycin and colistin respectively, and conjugating them with PAA to yield to a new chemical entity (PAA-VC) active itself toward both types of bacteria (Scheme 1). Because PAA-VC is conceived to target the entire bacterial surface layers by means of a broad range of destabilizing mechanisms (i.e., D-alanyl-D-alanine blocking, transmembrane pore formation, electrostatic outermost membrane perturbation), it might be expected that bacteria will develop resistance with great difficulty. The aim of colistin (C) and vancomycin (V) conjugation is manifold. Colistin, also known as polimixin E, is a branched polycationic and amphiphilic peptide produced by Paenibacillus

polymyxa

subspecies

colistinus

capable

of

strongly

interacting

with

lipopolysaccharide (LPS) in the outer cell membrane of Gram-negative bacteria by displacing divalent ions (i.e., Mg+2 and Ca+2) from the phosphate groups of the Lipid A,32–34 leading to disruption of the outer cell membrane. It performs a multiple perturbation throughout the outmost 13 ACS Paragon Plus Environment


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membrane limiting the development of resistant strains of bacteria.18 So, despite it was shelved in the past owing to nephrotoxicity and neurotoxicity,35 inhaled colistin36 and the reuse of intravenous colistin have been reconsidered for the treatment of serious MDR P. aeruginosa, A. baumannii and Enterobacteriaceae infections.37 On the other hand, vancomycin is a tricyclic glycosylated peptide containing aromatic moieties produced by Actinobactera species Amycolatopsis orientalis active against Gram-positive microorganisms. It binds the D-alanyl-D-alanine peptide preventing the crosslinking reaction of the bacterial cell wall precursors, thus inhibiting the synthesis of peptidoglycan in bacteria. It is considered the drug of choice in the treatment of Methicillin resistant Staphylococcus aureus (MRSA) and to prevent biofilm-associated infections established on indwelling devices such as catheters.7


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Scheme 1. Synthesis of PAA-VC. Poly(argilyl)aspartamide (PAA): α and β aspartic acid repeating units (orange) and arginine pendants (purple).

However, in recent times the use of larger doses of vancomycin aimed at curbing the increasing incidence of resistant strains of S. aureus has led to a wider report of acute kidney injury.38 Side effects provoked by both peptides are clearly due to their biodistribution in the body, especially after topic administration. Moreover, peptides are incline to be hydrolytically deactivated upon administration, leading to byproducts. It might be expected that their conjugation with a biocompatible high molecular weight PAA will improve their chemical stability and biodistribution 15 ACS Paragon Plus Environment


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thus reflecting on the antimicrobial effectiveness and pharmacological safety.39 Moreover, PAA is a polyelectrolyte endowed with mucoadhesive properties and, hence, capable of perpetrating the potential broad spectrum biocide actions of PAA-VC upon a topical administration, enhancing the overall efficacy and minimizing side effects.40

Synthesis of PAA-VC. A high molecular weight poly(L-argilylaspartamide-co-aspartic) acid 78:22 (PAA) with narrow distribution was firstly obtained under basic conditions (pH 10.5) to lead to a polypeptide containing one carboxyl group per each repeating unit counterbalanced by 0.78 guanidine functions (Scheme 1 and Table 1). In order to explore the possibility of imparting PAA with broad spectrum bactericidal activity, it was covalently conjugated with colistin and vancomycin, by coupling the carboxyl groups of the polymeric backbone with the amine groups of the two peptides (Scheme 1). In the first step, colistin was conjugated to PAA using EDC and NHS as activating agents at pH 6.8. Being colistin a five-arms polyamine, a slight excess colistin was employed to avoid the concurrent ramification. A conjugate sample with Mw = 65.200 and PD = 1.40 was obtained in 80% yield after purification. The presence of electrostatic aggregates between colistin and PAA was excluded by SEC analysis in a high saline eluent (0.1 M Tris buffer pH 8.10 ± 0.05 with 0.2 M NaCl) combining traces obtained by triple detector (LALS, RALS and RI), which gave no evidence of multiple peaks. The structural assessment of the conjugate and its functionalization degree was attained by 1H NMR spectrometry (Figure 1). There are five peculiar resonances within the range 4.0 - 4.75 ppm, corresponding in


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Figure 1. 1H NMR spectra of PAA-VC in DMF/D2O and PAA-C in D2O, pD 6.5.

turn to the methine resonance ascribable to the colistin side chain and the α and β aspartic acid/aspartamide units. The methylene protons of the aminoethyl moyieties of colistin (4H) are also clearly detectable at 2.15 and 2.25 ppm. The 1H NMR spectrum of PAA-C also allowed to establish the amount of colistin conjugated to the main backbone, calculated comparing the integrals of the methyl protons of colistin side chains (18H) observed at 0.82 ppm with the methylene hydrogens of the backbone (2H) noticed at 2.65 and 2.70 ppm, attributable to the aspartic acid and argilylaspartamide repeating units, respectively. (Table 1). 17 ACS Paragon Plus Environment


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Table 1. Chemical and physicochemical characteristics of the conjugate compared with the parent compound: weighted molecular weight Mw, polydispersity (Mw/Mn), derivativation degree (DD) and Zpotential (ζ) Sample

Mw# (kDa)

Mw/Mn#

Composition

ζ∗∗ (mV)

PAA

62.1

1.41

DDArg* (mol%) 78

DDVanco* DDColistin* (mol%) (mol%) -21.2±2.0

PAA-C

65.2

1.40

78

-

3.9

-13.4±0.9

PAA-VC

67.1

1.34

78

5.8

3.9

-10.9±1.0

#Calculated by SEC analysis in TRIS buffer pH 8.1 *Obtained by means of 1H NMR spectroscopy **Obtained in PBS pH 7.4

Vancomycin consists of a heptapeptide conjugated with a disaccharide containing the highly reactive deoxyaminosugar vancosamine, which is considered to have pharmacokinetic functions. On the contrary, the heptapeptide provides five hydrogen bonds responsible for the binding specificity displayed for D-alanyl-D-alanine and represents the pharmacophore moiety. The Nmodification of the vancosamine moiety has attracted great attention since results in a compound that

acts

via

a

multitarget

fashion

exhibiting

activity

against

vancomycin-resistant

microorganisms.41 Indeed, it has been found that sugar-modified vancomycin alters the polymerization of peptidoglycan at multiple levels by inhibiting the glycopeptide monomer, straight-chain polymer and cross-link reinforcement.42,43 Here we synthetized a vancomycincontaining polypeptide, named PAA-VC, by conjugating vancomycin via EDC/NHS coupling reaction between vancosamine and carboxyl groups of the PAA-C backbone above described (Scheme 1). Using this approach one runs the risk of causing random crosslinking reaction of the polypeptide as amines of colistin side chains are also targets of NHS-activated carboxyl groups. However, as for protein conjugation, it is possible to empirically circumvent this phenomenon 18 ACS Paragon Plus Environment

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carrying out the reaction in diluted regimen, where intermolecular reactions are kinetically denied. Under these conditions a PAA-VC sample with Mw = 67100 and a narrower polydispersity was obtained (Table 1).

Figure 2. SEC traces of PAA and PAA-VC obtained in 0.1 M Tris buffer pH 8.10 ± 0.05 with 0.2 M

sodium chloride using a RI detector. Intermolecular dimerization doubling the molecular weight was excluded analyzing SEC traces obtained for PAA-VC in comparison with the parent polypeptide (Figure 2). A mono-modal chromatogram with narrow molecular weight distribution was detected, accompanied by a left-shift of the curve in comparison with PAA, indicating that the molecular weight of PAA-VC proportionally increased after conjugation with colistin and vancomycin. The structural identification of the conjugate was achieved by 1H NMR spectrometry in DMF/D2O. A typical spectrum of PAA-VC is reported in Figure 1 and shows the expected signal of the aromatic groups (9H) of the aglycone at 6.4 – 7.5 ppm, together with characteristic signals of the vancosamine moiety at 3.70 ppm. The derivatization degree in vancomycin was calculated on a molar basis comparing the integrals of the aromatic protons at 7.31 ppm ascribable to vancomycin (5H) with the methylene protons of the arginine side groups (2H) noticed at 3.15 ppm. Results are reported in Table 1. On the basis of the average molecular weight of the repeating unit ( !" = 364.19), calculated using the derivatization degree obtained, each polymer chain contained ten vancomycincontaining repeating units, while the amount of colistin side chains was seven. 19 ACS Paragon Plus Environment


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Biophysical Characterization of the PAA-VC Derivative. A further confirmation of the efficient conjugation with both antimicrobial polypeptides was obtained by Z-potential analysis carried out in water. L-arginine is covalently linked to the backbone involving its amino group and the resulting zwitterion side function do not alter the charge of the polypeptide. Thereby, the aspartic acid moiety (about 22%) are responsible for the high negative charge of PAA, that bias its antimicrobial activity owing to electrostatic repulsions with the phospholipids of the bacterial membrane. The charge balance of this polypeptide is strongly affected by the conjugation with both colistin and vancomycin, being highly cationic in physiologic media. As expected, the Z-potential of the polypeptide decreased after conjugations, passing from -21.2 to -10.9, facilitating the interactions with the bacterial membrane, which have been shown to be important for arginine and peptides antimicrobial activity.

Chemical Stability of PAA-VC in PBS pH 7.4 and 37 °C. To evaluate stability of peptides upon conjugation, PAA-VC was incubated in physiological medium for 7 days at 37 °C and analyzed by HPLC using a diode UV detector reading within the range 190 – 400 nm. The retention time observed for PAA-VC at T0 was 3.51 min, with the maximum absorption at 190 nm. After 7 days incubation, the chromatogram of PAA-VC appears complex (Figure 3). In particular, two additional peaks are revealed at r.t. 3.77 and 4.79 min, with UV absorption profiles typical of the pristine colistin sulfate (Figure 3). This is a clear evidence that colistin-containing polymer chains (about 2% on a weight basis) are somehow cleaved and released to the medium. On the contrary, vancomycin moieties did not undergo significant hydrolysis since peaks with its peculiar UV absorption curve (190 – 305 nm) were not revealed (Figure 3). On the whole, these findings suggest that the conjugate is quite stable in physiological medium and capable of acting as it is, without releasing significant amount drugs.


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Figure 3. HPLC traces of PAA-VC obtained after 7 days incubation in PBS pH 7.4 at 37 °C compared with vancomycin and colistin sulfate standards Evaluation of the mucoadhesive properties of PAA-VC. We further investigated the ability of PAA-VC to preserve mucoadhesive properties of the parent polypeptide (PAA), implying obvious advantages in topical treatments. This is a trouble of particular interest especially to treat mucosal biofilm-related infections (especially pulmonary, vaginal, ocular and intestinal), where extended high amount of antibiotics is desired to eradicate chronic infections and avoid MDR. The 21 ACS Paragon Plus Environment


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interaction of PAA-VC with mucin, commonly employed as a measure of the adhesivity between the mucus layer and polymers,44 was assessed turbidimetrically in PBS pH 7.4 and results are reported in Figure 4. PAA-VC caused a sharp decrease in transmittance of a solution of mucin after few minutes and with a remarkable time-dependent trend, which is a measure of the formation of electrostatic aggregates between mucin and the conjugate. The equilibrium seemed to be reached after 300 minutes as suggested by the appearance of a plateau even at the highest concentration (200 µg mL-1). The formation of aggregates was also affected by the concentration of the conjugate. In particular, after an incubation time of 50 min the transmittance passed from 54 to 39 % for the sample containing 50 and 200 µg mL-1 of PAA-VC respectively.

Figure 4. Mucoadhesive properties of PAA-VC in PBS pH 7.4. UV transmittance, λ 495 nm, of a solution of mucin 1 mg mL-1 (solid square), mucin 1 mg mL-1 and PAA-VC 50 µg mL-1 (open circle), mucin 1 mg mL-1 and PAA-VC 200 µg mL-1 (solid circle) In summary, PAA-VC showed excellent mucoadhesive properties in physiological conditions potentially exploitable for the in situ treatment of mucosal infections. Cytocompatibility Assay. An important aspect in the evaluation of new antimicrobial agents is the assessment of its cytocompatibility both after acute and chronic exposure, which should permit 22 ACS Paragon Plus Environment

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prolonged treatments and high dosage. Furthermore, the therapeutic index (TI) of new antimicrobial drugs can be obtained on the basis of toxicity studies that are significant to compare the advantages to existing clinical approaches.

Figure 5. Cytocompatibility of PAA-VC on 16 HBE cell line after acute (a) and chronic exposure (b). a: PAA-VC (solid symbol), vancomycin (red fitting) and colistin sulfate (green fitting). b: 2



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(solid symbol) and 7 (open symbol) days incubation with PAA-VC. IC50 calculated at 48 hours incubation. Here, we compared the acute cytotoxicity of the conjugate and free drugs on Human Bronchial Epithelium cells (16 HBE), until a concentration of 1 mg mL-1 (Figure 5a). PAA-VC did not show any toxicity against 16 HBE up to the maximum tested concentration, while both free peptides displayed certain cytotoxicity at concentration higher than 100 µg mL-1. Moreover, in an experimental study of chronic exposure in vitro PAA-VC conjugate resulted not cytotoxic after seven days incubation at concentration up to 10 mg mL-1 (Figure 5b). For comparison purposes the IC50 values of PAA-VC and the free peptides are reported in Figure 5. Despite the fact that PAAVC has an IC50 about two times lower than that observed for vancomycin and colistin on the basis of molar concentration (87 vs 170 µM), this is about 45 times higher on a w/v concentration basis. This is just the result of the higher molecular weight of PAA-VC compared to the free peptides, which is expected to influence both IC50 and MIC values. Therefore, a more appropriate analytical evaluation of the potential therapeutic benefits of PAA-VC requires the therapeutic indices (LogTI) calculation as showed below (Table 2). However, compared with macromolecular antiseptics and disinfectants such as quaternary ammonium polyacrilates and vinyl polymers already reported (IC50 < 100 µg mL-1)45 the IC50 value of PAA-VC is much higher (about 145 times). Hence, the IC50 observed is foreseen to play an important role in reaching proper dosage without hinting at significant toxic effects. Antimicrobial activity. First of all, we compared the in vitro activity of the PAA-VC conjugate in inhibiting the growth of planktonic forms of clinical and reference strains of S. aureus and P. aeruginosa with the pristine colistin sulphate and vancomycin hydrochloride. These two pathogens were chosen since they are listed by the WHO as antibiotic-resistant “priority pathogens” that pose the greatest threat human health.2,23 In particular, the most critical group includes carbapenem-


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resistant Pseuudomonas aeruginosa and the high priority group includes methicillin-resistant Staphylococcus aureus. The activities in richer medium Tryptic Soy Broth (TSB) are reported in Table 2 in terms of MIC values in µg mL-1 and µM. As expected, colistin and vancomycin showed MICs values in the µM range only for P. aeruginosa and S. aureus strains respectively, whereas the conjugate was active against both tested microorganisms. In particular, PAA-VC was active toward both clinical strains and the

Table 2. In vitro antimicrobial activity against S. aureus and P. aeruginosa (planktonic forms) in terms of MIC values and Log therapeutic index (Log TI): P. aeruginosa 4 - metallo-beta-lactamase (M βL) producing, P. aeruginosa 4 - sensitive, S. aureus 69 - MRSA and S. aureus 62 - MSSA Microorganism

MIC Values PAA-VC µg ml

-1

Vancomycin µg ml

-1

(µM)

(µM)

P. aeruginosa ATCC 15442

3.125 (0.019)

> 200

P. aeruginosa 31 – sensitive

12.5 (0.075) 12.5 (0.075) 12.5 (0.075) 6.25 (0.037) 6.25 (0.037)

P. aeruginosa 4 - M βL S. aureus ATCC 25923 S. aureus 69 – MRSA S. aureus 62 – MSSA

> 200 > 200 1.6 (1.07) 3.125 (2.14) 6.25 (4.28)

Log TI Colistin

PAA-VC

Vancomycin

Colistin

3.67

n.a.

2.43

3.06

n.a.

2.13

3.06

n.a.

2.13

> 200

3.06

2.30

n.a.

> 200

3.37

2.01

n.a.

> 200

3.37

1.71

n.a.

µg ml

-1

(µM)

0.8 (0.63) 1.6 (1.26) 1.6 (1.26)

reference strain of S. aureus at a MIC concentration from 10- to 48-fold lower on a molar basis (0.09 µM vs 4.28 µM for PAA-VC and vancomycin respectively). A similar trend was observed for P. aeruginosa strains. The exposure to PAA-VC resulted in a more than 10-fold decrease in MICs 25 ACS Paragon Plus Environment


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(0.048 µM vs 0.63 µM for PAA-VC and colistin respectively) (see Table 2). These results are very interesting

also

if

compared

with

others

macromolecular

polycations

such

as

hydroxypropyltrimethyl ammonium chloride chitosan (HACC), where MICs values observed for MRSA are 400-fold higher on a weight basis (6.25 µg mL-1 vs 2.5 mg mL-1).46 The therapeutic indices (LogTI) of PAA-VC and the parent compounds are reported in Table 2 to assess the pharmacological versatility of PAA-VC and to compare the antimicrobial efficacy with the pristine side chains (vancomycin and colistin). The LogTI calculated for PAA-VC were significantly advantageous with respect to both pristine drugs for all microorganism considered, revealing that a synergistic antimicrobial effect between arginine side groups and the conjugated antimicrobial peptides occurred. In addition, the LogTI observed for PAA-VC is 2-fold higher of that observed for recent antimicrobial peptide-mimetic methacrylates polymers (3.6 vs 1.5).3 Preliminary tests carried out using the physical mixture of both pristine colistin and vancomycin corroborated this interpretation since their MICs never seemed to relate to the combination of both drugs (Supporting information, Table S2, Pag. S-4). It might be noticed that despite the high molecular weight of PAA-VC and the considerable chemical modification of both antimicrobial peptides side chains (i.e., vancomycin and colistin), the ability to perturb the bacterial membrane and wall was preserved, giving rise to a broad spectrum of activity. We could explain this behavior by the random coil distribution of the PAA-VC side chains, which allows both pendants to be available to interact with the bacterial wall and/or membrane, and to the cationic features of PAA-VC provided by the colistin repeating units. Although the detailed mechanism is still largely unknown, it is plausible that PAA-VC chains engage the loosely packed peptidoglycan layer facilitating deep penetration of the vancomycin pendants inside the cell to interact with the cytoplasmic membrane through colistin and guanidine side chains.47 The cooperative action of the bioactive side chains might explain the enhanced biocidal activity observed for the conjugate.

Concerning the activity against the growth as sessile community (biofilm), we compared the ability to inhibit the formation of a biofilm after 24 h using MIC and sub-MIC concentrations of PAA-VC 26 ACS Paragon Plus Environment

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or free antibiotics, considering the MIC values obtained against planktonic forms (Figure 6). The conjugate inhibited significantly the biofilm formation of both S. aureus and P. aeruginosa strains at MIC values. In particular, we reported percentages of inhibition of 85.8% at 6.25 µg mL-1 (0.15 µM) toward S. aureus 62, 70.6% for S. aureus 69 and 74.7% for S. aureus ATCC 25923. For all S. aureus, except for reference strain, no sub-MIC values were effective in biofilm inhibition formation.



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Figure 6. Inhibition of staphylococcal (a) and pseudomonal (b) biofilm formation: comparison between PAA-VC, vancomycin hydrochloride, and colistin sulfate Regarding P. aeruginosa, we observed percentage of biofilm inhibition of 75.2% for P. aeruginosa 31, 71.2% for P. aeruginosa 4 and 46.7% for P. aeruginosa ATCC 15442. For clinical and reference strains of P. aeruginosa, we noticed a very moderate biofilm inhibition at sub-MIC values with percentages ranged from 5 to 13.5%. Vancomycin and colistin sulphate, comparatively tested, can inhibit significantly biofilm formation respectively of S. aureus or P. aeruginosa at MIC values with percentages of 71.3% for S. aureus 62, 78.3% for S. aureus 69, 88.6% for S. aureus ATCC 25923, 34.0% for P. aeruginosa 31. No inhibition of biofilm formation was recorded at MIC values for other strains of P. aeruginosa. We also tested the anti-biofilm activity of PAA-VC against a mature staphylococcal or pseudomonal biofilm 24 hours-old, after 24-h exposure at different concentrations equal or greater than MICs obtained against planktonic strains (500, 100, 50, 12.5 and 6.25 µg mL-1). For clinical S. aureus we reported a mean percentage of biofilm reduction of 83.4% and of 90.5% for reference 28 ACS Paragon Plus Environment

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strain (Figure 7a); significantly improved if compared with the free drug at concentration about 20fold higher (2.9 vs 69 µM). Besides, for P. aeruginosa, we observed a relevant percentage of biofilm reduction for clinical strains (89.3% for metallo-ß-lactamse producing strain and 68.0% for sensitive strain) and for reference ATCC (81,2%) as well (Figure 7b). Moreover, for PAA-VC a biofilm reduction of 0.6%, 25.5% and 35.5% occurred at MIC values for S. aureus 62 S. aureus 69 and S. aureus ATCC 25923, respectively (Figure 7a). A similar trend was observed for the free vancomycin. On the contrary, pseudomonal biofilms underwent only a slight inhibition if treated with colistin at MIC values (from 5.2 to 7.5 % for reference and clinical strain respectively), as an inhibition up to 29.4% was observed for the conjugate at the MIC values (Figure 7b).



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Figure 7. Anti-biofilm activity against staphylococcal (a) and pseudomonal (b) biofilms 24 h old: comparison between PAA-VC, vancomycin hydrochloride and colistin sulfate In general, notwithstanding the high molecular weight of PAA-VC (167 kDa), which might negatively affect the efficacy on Gram-negative bacteria such as P. aeruginosa owing to various factors related to entering the outer membrane,48 best results were obtained with the conjugate in comparison with the free drugs. The conjugate showed a significant biofilm removal for all microorganisms tested at concentration of 500 µg mL-1 (2.99 µM), displaying broad spectrum activity toward clinically isolated strains (Figure 7). Noteworthy is that, thanks to the amphoteric nature of PAA-VC and the consequent high biocompatibility (IC50 on 16 HBE = 14.5 mg mL-1), PAA-VC could be employed at higher dosage compared with the parent drugs therefore providing a good therapeutic window in view of clinical applications. The ability of the PAA-VC conjugate to kill established biofilms was also confirmed by live⁄dead staining on S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 biofilms in combination with confocal microscopy. This provide additional information on the status of individual cells within the 30 ACS Paragon Plus Environment

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biofilm and the effect of treatments on cell viability. Either S. aureus ATCC 25923 or P. aeruginosa ATCC 15442 were grown on the surface of the bottom glass slide of a static chamber for 24 hours. Both staphylococcal and pseudomonal reference strains were exposed to PAA-VC or the reference drugs at concentration of 50 µg mL-1. Characteristic images of each treatment is reported in Figure 8. Overall, according to crystal violet tests, the established biofilm mass decreased after the treatment both with the reference drugs and the conjugate. Moreover, When the sessile community of both microorganisms was treated with PAA-VC it was observed a strong reduction of the biofilm viability and population (Figure 8). Such decrease is more evident than that one obtained by testing the free reference antibiotics. This is shown in Figure 8 where the PAA-VC treated cells displayed a feeble green florescence (live cells) accompanied with an extended red fluorescence (death cells), compared to the colistin and vancomycin treated control. Only small live colonies were detected following treatment with PAA-VC, indicating enhanced broad spectrum antimicrobial activity toward established biofilms.



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Figure 8. Confocal microscopy images of S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 biofilms treated with PAA-VC, vancomycin and colistin obtained by live/dead staining. Syto9 (green; viable cells) and propidium iodine (red; dead cells) stained images were acquired at 60x magnification. CONCLUSIONS To circumvent the irregular release issues of release-based antimicrobial biomaterials, a semisynthetic branched high molecular weight glyco-polypeptide with inherent bactericidal activity, named PAA-VC, that consists of a polyaspartic acid backbone functionalized with L-arginine, vancomycin and colistin side chains, has been developed. This glyco-polypeptide is stable under physiological conditions and effective against both Gram-positive and Gram-negative bacteria such 32 ACS Paragon Plus Environment

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as Staphylococcus aureus and Pseudomonas aeruginosa at nM concentrations (90 and 190 nM, respectively). We found that the conjugation of colistin and vancomycin with a synthetic argininerich polyampholyte results in an optimization of the antimicrobial properties in terms of broadspectrum antimicrobial activity and therapeutic indices (3.06 < Log TI < 3.67), allowing to potentially address empiric treatment for severe infections in critical patients. PAA-VC also showed negligible cytotoxicity on 16 HBE cells with an IC50 about 45 times (i.e., 14.5 mg mL-1 vs 250 µg mL-1) higher than that observed for the most popular quaternary ammonium compounds reported elsewhere. Notable properties of PAA-VC include mucoadhesive ability exploitable for improving bioavailability after topical administration (e.g., against pulmonary and vaginal infections). Most importantly, as peptides and antibiotics are poorly effective against biofilms we found that PAA-VC is able to both avoid biofilm formation and kill established biofilms of two relevant pathogens at micromolar concentration (94 and 82 % biofilm break-up for S. aureus and P. aeruginosa, respectively). Overall, the unusual antimicrobial properties of PAA-VC and its high cytocompatibility, together with the versatile synthetic approach employed, make it a promising candidate for potential preclinical studies on eradicating severe infections using in vivo animal models. The availability of a wide spectrum therapeutic molecule against both Gram positive and Gram negative bacteria would be an important resource for treatment of severe infections in critical patients and requires further studies to better investigate possible clinical applications. ASSOCIATED CONTENT Supporting Information. 13C NMR spectra of PAA and PAA-VC, Minimum Inhibitory Concentrations (MICs) in MH, MH2 and TSB. MICs of the mixture of reference antibiotics and PAA.

AUTHOR INFORMATION Corresponding Author [email protected]. FAX: +39 09123891928 Tel: +39 09123891928. Author Contributions 33 ACS Paragon Plus Environment


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N.M. conceived and managed the project, synthesized the conjugate, performed and analyzed all chemical and physicochemical characterizations, performed cytocompatibility tests and wrote the main manuscript. D.S. conceived the microbiological tests and contributed to write the manuscript. P.V. contributed to synthetize the conjugates and helped to perform chemical and physicochemical experiments. M.G.C. and D.M.G. performed the microbiological characterizations and analyzed the results. M.G. conceived the project from the clinical point of view. G.C. revised all Figures and Tables and helped to write the manuscript. C.M.M. contributed to conceive the microbiological tests and contributed to write the manuscript. G.G. contributed to manage the project and revised the final manuscript. All authors reviewed and approved the final manuscript.

Notes The authors declare no competing financial interest. REFERENCES

(1)

Gould, I. M.; Bal, A. M. New Antibiotic Agents in the Pipeline and How They Can Help Overcome Microbial Resistance. Virulence 2013, 4 (2), 185–191.

(2)

Willyard, C. The Drug-Resistant Bacteria That Pose the Greatest Health Threats. Nat. News 2017, 543 (7643), 15.

(3)

Takahashi, H.; Nadres, E. T.; Kuroda, K. Cationic Amphiphilic Polymers with Antimicrobial Activity for Oral Care Applications: Eradication of S. Mutans Biofilm. Biomacromolecules 2017, 18 (1), 257–265.

(4)

Centers for Disease Control and Prevention (US). Antibiotic Resistance Threats in the United States, 2013. 2013.

(5)

Rossolini, G. M.; Arena, F.; Pecile, P.; Pollini, S. Update on the Antibiotic Resistance Crisis. Curr. Opin. Pharmacol. 2014, 18, 56–60.

(6)

Hengzhuang, W.; Wu, H.; Ciofu, O.; Song, Z.; Høiby, N. Pharmacokinetics/pharmacodynamics of Colistin and Imipenem on Mucoid and Nonmucoid


Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pseudomonas Aeruginosa Biofilms. Antimicrob. Agents Chemother. 2011, 55 (9), 4469– 4474. (7)

Bjarnsholt, T.; Ciofu, O.; Molin, S.; Givskov, M.; Høiby, N. Applying Insights from Biofilm Biology to Drug Development - Can a New Approach Be Developed? Nat. Rev. Drug Discov. 2013, 12 (10), 791–808.

(8)

Doernberg, S. B.; Chambers, H. F. Antimicrobial Stewardship Approaches in the Intensive Care Unit. Infectious Disease Clinics of North America. Elsevier September 1, 2017, pp 513– 534.

(9)

Kenawy, E.-R.; Worley, S. D.; Brouhton, R. The Chemistry and Applications of Antimicrobial Polymers: A State-of-the-Art Review. Biomacromolecules 2007, 8 (5), 1359– 1384.

(10)

Jain, A.; Duvvuri, L. S.; Farah, S.; Beyth, N.; Domb, A. J.; Khan, W. Antimicrobial Polymers. Adv. Healthc. Mater. 2014, 3 (12), 1969–1985.

(11)

Nguyen, T.-K.; Lam, S. J.; Ho, K. K.; Kumar, N.; Qiao, G. G.; Egan, S.; Boyer, C.; Wong, E. H. Rational Design of Single-Chain Polymeric Nanoparticles That Kill Planktonic and Biofilm Bacteria. ACS Infect. Dis. 2017, 3 (3), 237–248.

(12)

Wang, L.-S.; Gupta, A.; Rotello, V. M. Nanomaterials for the Treatment of Bacterial Biofilms. ACS Infect. Dis. 2016, 2 (1), 3–4.

(13)

Ferruti, P.; Mauro, N.; Falciola, L.; Pifferi, V.; Bartoli, C.; Gazzarri, M.; Chiellini, F.; Ranucci, E. Amphoteric, Prevailingly Cationic L -Arginine Polymers of Poly(amidoamino Acid) Structure: Synthesis, Acid/Base Properties and Preliminary Cytocompatibility and Cell-Permeating Characterizations. Macromol. Biosci. 2014, 14 (3), 390–400.

(14)

Licciardi, M.; Li Volsi, A.; Sardo, C.; Mauro, N.; Cavallaro, G.; Giammona, G. InulinEthylenediamine Coated SPIONs Magnetoplexes: A Promising Tool for Improving siRNA Delivery. Pharm. Res. 2015, 32 (11), 3674–3687.

(15)

Chan, D. I.; Prenner, E. J.; Vogel, H. J. Tryptophan- and Arginine-Rich Antimicrobial 35 ACS Paragon Plus Environment


Page 36 of 40

Peptides: Structures and Mechanisms of Action. Biochim. Biophys. Acta - Biomembr. 2006, 1758 (9), 1184–1202. (16)

Strom, M.; Haug, B. E.; Rekdal, O.; Skar, M.; Stensen, W.; Svendsen, J. Important Structural Features of 15-Residue Lactoferricin Derivatives and Methods for Improvement of Antimicrobial Activity. Biochem. Cell Biol. 2002, 80 (1), 65–74.

(17)

Griffiths, P. C.; Mauro, N.; Murphy, D. M.; Carter, E.; Richardson, S. C. W.; Dyer, P.; Ferruti, P. Self-Assembled PAA-Based Nanoparticles as Potential Gene and Protein Delivery Systems. Macromol. Biosci. 2013, 13 (5), 641–649.

(18)

Brogden, K. A. Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria? Nat. Rev. Microbiol. 2005, 3 (3), 238–250.

(19)

Bradshaw, J. P. Cationic Antimicrobial Peptides. BioDrugs 2003, 17 (4), 233–240.

(20)

Mauro, N.; Fiorica, C.; Varvarà, P.; Di Prima, G.; Giammona, G. A Facile Way to Build up Branched High Functional Polyaminoacids with Tunable Physicochemical and Biological Properties. Eur. Polym. J. 2016, 77, 124–138.

(21)

Aliste, M. P.; MacCallum, J. L.; Tieleman, D. P. Molecular Dynamics Simulations of Pentapeptides at Interfaces: Salt Bridge and Cation-Pi Interactions. Biochemistry 2003, 42 (30), 8976–8987.

(22)

Chen, L.; Chen, T.; Fang, W.; Wen, Y.; Lin, S.; Lin, J.; Cai, C. Synthesis and pH-Responsive “Schizophrenic” Aggregation of a Linear-Dendron-Like Polyampholyte Based on Oppositely Charged Polypeptides. Biomacromolecules 2013, 14 (12), 4320–4330.

(23)

World Health Organization. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. 2017.

(24)

Giammona, G.; Carlisi, B.; Palazzo, S. Reaction of Α,β-poly(N-Hydroxyethyl)-DLAspartamide with Derivatives of Carboxylic Acids. J. Polym. Sci. Part A Polym. Chem. 1987, 25 (10), 2813–2818.

(25)

Ferruti, P.; Mauro, N.; Manfredi, A.; Ranucci, E. Hetero-Difunctional Dimers as Building 36 ACS Paragon Plus Environment

Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Blocks for the Synthesis of Poly(amidoamine)s with Hetero-Difunctional Chain Terminals and Their Derivatives. J. Polym. Sci. Part A Polym. Chem. 2012, 50 (23), 4947–4957. (26)

Fiorica, C.; Mauro, N.; Pitarresi, G.; Scialabba, C.; Palumbo, F. S.; Giammona, G. DoubleNetwork-Structured Graphene Oxide-Containing Nanogels as Photothermal Agents for the Treatment of Colorectal Cancer. Biomacromolecules 2017, 18 (3), 1010–1018.

(27)

Raimondi, M. V.; Maggio, B.; Raffa, D.; Plescia, F.; Cascioferro, S.; Cancemi, G.; Schillaci, D.; Cusimano, M. G.; Vitale, M.; Daidone, G. Synthesis and Anti-Staphylococcal Activity of New 4-Diazopyrazole Derivatives. Eur. J. Med. Chem. 2012, 58, 64–71.

(28)

O’Toole, G. A. Microtiter Dish Biofilm Formation Assay. J. Vis. Exp. 2011, No. 47, 2437.

(29)

Schillaci, D.; Arizza, V.; Dayton, T.; Camarda, L.; Di Stefano, V. In Vitro Anti-Biofilm Activity of Boswellia Spp. Oleogum Resin Essential Oils. Lett. Appl. Microbiol. 2008, 47 (5), 433–438.

(30)

Tonna, N.; Bianco, F.; Matteoli, M.; Cagnoli, C.; Antonucci, F.; Manfredi, A.; Mauro, N.; Ranucci, E.; Ferruti, P. A Soluble Biocompatible Guanidine-Containing Polyamidoamine as Promoter of Primary Brain Cell Adhesion and in Vitro Cell Culturing. Sci. Technol. Adv. Mater. 2014, 15 (4), 45007.

(31)

Kenawy, E.-R.; Abdel-Hay, F. I.; El-Shanshoury, A. E.-R. R.; El-Newehy, M. H. Biologically Active Polymers. V. Synthesis and Antimicrobial Activity of Modified Poly(glycidyl Methacrylate-Co-2-Hydroxyethyl Methacrylate) Derivatives with Quaternary Ammonium and Phosphonium Salts. J. Polym. Sci. Part A Polym. Chem. 2002, 40 (14), 2384–2393.

(32)

Sakura, N.; Itoh, T.; Uchida, Y.; Ohki, K.; Okimura, K.; Chiba, K.; Sato, Y.; Sawanishi, H. The Contribution of the N-Terminal Structure of Polymyxin B Peptides to Antimicrobial and Lipopolysaccharide Binding Activity. Bull. Chem. Soc. Jpn. 2004, 77 (10), 1915–1924.

(33)

Liu, Y. Y.; Wang, Y.; Walsh, T. R.; Yi, L. X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; Yu, L. F.; Gu, D.; Ren, H.; Chen, X.; Lv, L.; He, D.; Zhou, H.; Liang, 37 ACS Paragon Plus Environment


Page 38 of 40

Z.; Liu, J. H.; Shen, J. Emergence of Plasmid-Mediated Colistin Resistance Mechanism MCR-1 in Animals and Human Beings in China: A Microbiological and Molecular Biological Study. Lancet Infect. Dis. 2016, 16 (2), 161–168. (34)

Groisman, E. A.; Kayser, J.; Soncini, F. C. Regulation of Polymyxin Resistance and Adaptation to Low-Mg2+ Environments. J. Bacteriol. 1997, 179 (22), 7040–7045.

(35)

Biswas, S.; Brunel, J.-M.; Dubus, J.-C.; Reynaud-Gaubert, M.; Rolain, J.-M. Colistin: An Update on the Antibiotic of the 21st Century. Expert Rev. Anti. Infect. Ther. 2012, 10 (8), 917–934.

(36)

Tumbarello, M.; De Pascale, G.; Trecarichi, E. M.; De Martino, S.; Bello, G.; Maviglia, R.; Spanu, T.; Antonelli, M. Effect of Aerosolized Colistin as Adjunctive Treatment on the Outcomes of Microbiologically Documented Ventilator-Associated Pneumonia Caused by Colistin-Only Susceptible Gram-Negative Bacteria. Chest 2013, 144 (6), 1768–1775.

(37)

Falagas, M. E.; Kasiakou, S. K. Colistin: The Revival of Polymyxins for the Management of Multidrug-Resistant Gram-Negative Bacterial Infections. Clin. Infect. Dis. 2005, 40 (9), 1333–1341.

(38)

Bamgbola, O. Review of Vancomycin-Induced Renal Toxicity: An Update. Ther. Adv. Endocrinol. Metab. 2016, 7 (3), 136–147.

(39)

Qi, Y.; Chilkoti, A. Protein–polymer Conjugation—moving beyond PEGylation. Curr. Opin. Chem. Biol. 2015, 28, 181–193.

(40)

Bruno, B. J.; Miller, G. D.; Lim, C. S. Basics and Recent Advances in Peptide and Protein Drug Delivery. Ther. Deliv. 2013, 4 (11), 1443–1467.

(41)

Ge, M.; Chen, Z.; Onishi, H. R.; Kohler, J.; Silver, L. L.; Kerns, R.; Fukuzawa, S.; Thompson, C.; Kahne, D. Vancomycin Derivatives That Inhibit Peptidoglycan Biosynthesis without Binding D-Ala-D-Ala. Science (80-. ). 1999, 284 (5413), 507–511.

(42)

Williams, D. H. The Glycopeptide Story--How to Kill the Deadly “Superbugs”. Nat. Prod. Rep. 1996, 13 (6), 469–477. 38 ACS Paragon Plus Environment

Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(43)

Křen, V.; Řezanka, T. Sweet Antibiotics – the Role of Glycosidic Residues in Antibiotic and Antitumor Activity and Their Randomization. FEMS Microbiol. Rev. 2008, 32 (5), 858–889.

(44)

Thongborisute, J.; Takeuchi, H. Evaluation of Mucoadhesiveness of Polymers by BIACORE Method and Mucin-Particle Method. Int. J. Pharm. 2008, 354 (1–2), 204–209.

(45)

Jiao, Y.; Niu, L. na; Ma, S.; Li, J.; Tay, F. R.; Chen, J. hua. Quaternary Ammonium-Based Biomedical Materials: State-of-the-Art, Toxicological Aspects and Antimicrobial Resistance. Progress in Polymer Science. Pergamon August 1, 2017, pp 53–90.

(46)

Peng, Z. X.; Wang, L.; Du, L.; Guo, S. R.; Wang, X. Q.; Tang, T. T. Adjustment of the Antibacterial Activity and Biocompatibility of Hydroxypropyltrimethyl Ammonium Chloride Chitosan by Varying the Degree of Substitution of Quaternary Ammonium. Carbohydr. Polym. 2010, 81 (2), 275–283.

(47)

Weber, D. J.; Rutala, W. A. Self-Disinfecting Surfaces: Review of Current Methodologies and Future Prospects. Am. J. Infect. Control 2013, 41 (5), S31–S35.

(48)

Lambert, P. A. Cellular Impermeability and Uptake of Biocides and Antibiotics in GramPositive Bacteria and Mycobacteria. J. Appl. Microbiol. 2002, 92 (s1), 46S–54S.



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Branched High Molecular Weight Glycopolypeptide With Broad

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