Lipase-Responsive Electrospun Theranostic Wound Dressing for

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Lipase-Responsive Electrospun Theranostic Wound Dressing for Simultaneous Recognition and Treatment of Wound Infection Hardev Singh, Wei Li, Mohammad Reza Kazemian, Runqiang Yang, Chengbo Yang, Sarvesh Logsetty, and Song Liu ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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ACS Applied Bio Materials

Lipase-Responsive Electrospun Theranostic Wound Dressing for Simultaneous Recognition and Treatment of Wound Infection Hardev Singha, Wei Lia, Mohammad Reza Kazemiana,b, Runqiang Yangc,d, Chengbo Yangc, Sarvesh Logsettye, and Song Liu*,a,b aDepartment

of Biosystems Engineering, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba

bBiomedical

Engineering, Faculty of Engineering, University of Manitoba, Winnipeg, Manitoba

cDepartment

dCollege

of Animal Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2

of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People's Republic of China

eDepartment

of Surgery and Psychiatry, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Canada

KEYWORDS: theranostic, smart wound dressing, lipase-responsive, chromogenic, ciprofloxacin

ABSTRACT Simultaneous monitoring and treatment of wound infection is of great importance in the biomedical field. The present work describes the development of a theranostic wound dressing (TH-WD) that can monitor and inhibit wound infection simultaneously. The main component of TH-WD is a polyurethane (PU) scaffold loaded with a ciprofloxacin-based prodrug (Pro-Cip) and 1 ACS Paragon Plus Environment

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a chromogenic probe (H-Cy). In vitro studies demonstrated that the TH-WD displayed efficient inactivation (100±4% reduction) of Pseudomonas aeruginosa (ATCC 27853) within 4 hours of contact whilst providing a visual detection of wound infection via a simple color change from yellow to green to red. These results are attributed to the activation of H-Cy and Pro-Cip via hydrolysis of their ester linkages catalyzed by lipase, an extracellular enzyme secreted by bacteria. Moreover, TH-WD is highly selective as it only changes color and releases the active drug (ciprofloxacin) in the presence of certain lipase-secreting pathogenic bacteria such as P. aeruginosa ATCC 27853 and no color change and cytotoxicity were observed when TH-WD was incubated with no- or low-lipase producing bacteria (e.g. E. coli TOP 10) or skin cell fibroblast. This hence can minimize the emergence of bacterial resistance associated with the overuse of antibiotics and avoid unnecessary cytotoxicity to skin cells. The present system not only provides a visible and non-invasive method to monitor the wound status but also allows the timely administration of antibacterial agents to inactivate bacteria in the wound.

1. INTRODUCTION Wound infections, typically characterized by the colonization of a wound by pathogenic organisms from the surrounding environment, have become a serious health problem for patients with open wounds. The wound offers these pathogens an environment in which they can nourish and proliferate, ultimately causing significant damage to host tissue. Wound infections play an important role in the development of chronic non-healing wounds that are a silent epidemic that affects a large portion of the population and often causes life threatening complications.1 For example, in the United States of America alone, about 1-2% of the population is estimated to be affected by chronic wounds causing morbidity in around 6.5 million patients at a cost of $25 billion

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per year.2 Normally, wounds are considered infected when the pathogenicity and virulence factors3 expressed by micro-organisms in the wound bed dominate the host’s natural immune response. Infection is characterized by clinical signs such as pain and redness, tenderness, erythema, heat, edema, and abscess/pus.4,5,6 Under clinical conditions, a constant and qualitative examination of wounds and patients for those signs is necessary. However, due to the variable presentation profiles, clinical assessment is not always reliable. Another clinical practice is to swab the wound site and test the swab in a microbiology lab for bacterial growth. Unfortunately, this method can be painful and time consuming. Since growth and identification of pathogenic bacteria in lab may take days or weeks to complete, the time required for the commencement of the management plan may be lengthy and require multiple samples, ultimately leading to increased costs and delays in treatment. This situation can become even more critical for chronic wound management since it has been shown that the longer the delay in treatment, the more difficult for a wound to fully heal.7,8 The prolonged healing is both a social and economic burden on the patient. Therefore, there is a need for more efficient detection methods that are quick, non-invasive, and more importantly reduce the delay between the identification of infection and its treatment. In response to this issue, advancements in the field of biomaterials have led to fabrication of ‘smart’ wound dressings. These dressings change their properties in response to the unique microenvironment of the wound itself and hence can alert medical staff of the possible presence of an infection without requiring the removal of the dressing, which can be very painful. Various efforts in this direction have been achieved through establishing a visible response to biochemical and/or physical markers expressed during wound infection.9 For example, Jenkins et. al.10 from the University of Bath unveiled a smart color-changing bandage that releases a non-toxic fluorescent dye in response to pathogenic bacteria. Similarly, a team from the University of Rochester has



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fabricated a bacteria-responsive porous silicon microcavity biosensor.11 The sensor was surface modified with tetratryptophan ter-cyclo pentane (TWTCP) receptors to target Lipid A, one of the endotoxins of Gram-negative bacteria. Binding between TWTCP and Lipid A leads to a bathochromic shift in the photoluminescence peak of the biosensor, which is read as a sign of bacterial infection. Liu et. al.12 and Kassal et. al.13 have respectively reported alginate- and polyurethane-based hydrogels incorporating pH-responsive dye as smart dressings for colorimetric sensing of the wound’s pH to detect infection. In a similar fashion, Schaude et. al.14 developed smart cotton swabs covalently modified with a pH-indicator for monitoring the pH of wounds. Another smart bandage developed by Kassal et. al.15 was employed for the determination of uric acid concentration, an important and specific biomarker of wound status. Most of these articles focus on detection/diagnosis of wound infections. Moving one step forward, we believe that the development of a smart theranostic wound dressing that not only allows the visual detection of infection but is also capable of releasing an antibacterial agent in response to wound infections is essential. ‘‘Theranostic’’ is a term used for describing the combination of diagnosis and therapy. A shift toward smart theranostic approaches contributes to the transition from conventional medicine to precision medicine. The main focus of this platform is to improve the pharmacokinetics and systematic administration of therapeutics to the site of action, thereby minimize the off-target effects of drugs. Although a smart theranostic approach has been explored extensively in cancer research, its applications in the field of microbiology have been limited. Specifically, there have been only few reports on theranostic wound dressings.16,17,18 We proposed to employ a prodrug strategy in the fabrication of a new smart theranostic wound dressing. A prodrug refers to a pharmacologically inactive compound that is transformed to an active substance through chemical or metabolic changes. This strategy has gained interest in the 4 ACS Paragon Plus Environment

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field of medicine such that nowadays approximately 10% of medicines used in therapy are administrated as prodrugs.19 This strategy not only avoids unwanted properties of the parent drug, but also helps control the concentration of the active drug. In the context of a wound, this means that the active drug will only be initiated when in the presence of bacteria, but otherwise remain as a dormant compound. Also, we adopted a chromogenic approach for the detection of bacterial infection. In this approach, smart membrane showed drastic color change in response to bacterial infection. This approach is non-invasive, straightforward, and offers a continuous monitoring of the wound environment without the need for complex equipment. The new smart theranostic wound dressing (TH-WD) was fabricated by blend electrospinning of polyurethane (PU, main scaffold) and enzyme-responsive small organic molecules (i.e., prodrug (Pro-Cip) and chromogenic probe (H-Cy)). The chromogenic probe, H-Cy (an ester derivative of a hemi-cyanine dye), was employed for colorimetric detection of bacterial infections since the dye is a well-known chromophore exhibiting strong intramolecular charge transfer (ICT) due to its electron “push– pull” substituents. Pro-Cip is a prodrug of Ciprofloxacin, a well-known antibiotic used for antibacterial applications. The in vitro studies demonstrated that the TH-WD displayed efficient killing/inhibition of Pseudomonas aeruginosa (ATCC 27853) whilst providing a visual detection of wound infection via a simple color change from yellow to green to red. These results are attributed to the activation of H-Cy and Pro-Cip via lipase-mediated hydrolysis of the ester linkages. Moreover, TH-WD is highly selective as it responds only when pathogenic bacteria exist. Essentially, it helps to further reduce the off-target effects of antibiotics and hence minimize the emergence of bacterial resistance associated with antibiotic overuse. The present system not only provides detection of wound infections in a non-invasive manner without removing the dressing



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but also allows the timely administration of an effective antibacterial agent to inhibit pathogen growth. 2. EXPERIMENTAL DETAIL 2.1. Materials and Chemicals 3-Hydroxy-3-methylbutan-2-one (95%), malonitrile (99%), 4-hydroxybenzaldehyde (98%), pyridine

(Py),

acetic

acid

(AcOH),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride (EDC) (98%), 4-(dimethylamino)pyridine (DMAP) (99%), ciprofloxacin (Cip) (98%),

boc-anhydride,

hexafluorophosphate

(HBTU)

& & &' &'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium (98%),

N,N-diisopropylethylamine

(DIPEA)

(99%),

6-

bromohexanol (97%), trifluoroacetic acid (TFA), polysuccinimide (PSI), propargyl amine, anhydrous N,N-dimethylformamide (DMF) (99%), dimethyl sulfoxide (DMSO) (99.5%), diethyl ether (99%), and tetrahydrofuran (THF) were purchased from Sigma Aldrich. 6-Bromohexanoic acid (98%), sodium ascorbate (99%), copper(II) sulfate pentahydrate (CuSO4·5H2O) were purchased from Alfa Aesar. The PU was hydrophilic aliphatic PU Tecophilic HP-60D-35 (PU60D) which was purchased from Lubrizol Advanced Materials (Cleveland, OH, USA). All chemicals used in the research were obtained from commercial sources as analytical reagents without further purification. 2.2. Synthetic Procedures The compounds 120, 221, 322 and Boc-protected ciprofloxacin (Boc-Cip)23 as shown in Scheme 1 were synthesized by following the known procedures while synthetic procedures for derivative 4 and final probes H-Cy and Pro-Cip are described below.


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Synthesis of H-Cy: A mixture of 6-azidohexanoic acid 2 (0.064 g, 0.41 mmol), EDCI (0.078 g, 0.41 mmol) and DMAP (0.05 g, 0.41 mmol) in 15 ml dry DCM was stirred under nitrogen at room temperature for 15 minutes. The hydroxyl derivative 1 (0.10 g, 0.33 mmol) was then added and stirring was continued overnight at room temperature. Upon completion of the reaction, DCM was evaporated to get the crude which was purified by column chromatography using 25% EtOAc/hexane to yield H-Cy in 56% yield. 1H NMR (DMSO-d6, 300 MHz) L 7.72-7.65 (m, 3H), 7.29 (d, J = 2.4 Hz, 2H), 7.02 (d, J = 16.5 Hz, 1H), 3.36 (t, J = 6.5 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.84 (s, 6H), 1.79-1.67 (m, 3H), 1.61-1.53 (m, 3H); 13C NMR (DMSO-d6, 75 MHz) L 175.37, 173.94, 171.75, 154.19, 146.34, 131.31, 130.38, 122.91, 114.93, 111.68, 110.96, 110.22, 100.11, 97.99, 97.97, 57.83, 34.36, 31.23, 26.41, 24.50, 22.33, 13.95; MS (ESI): m/z calcd. for C24H22N6O3: 442.1753 Found: 441.1732 [M-1]+. Synthesis of Pro-Cip: Boc-Cip (0.10 g, 0.23 mmol), HBTU (0.13 g, 0.35 mmol), and catalytic amount of DMAP were dissolved in 20 ml dry DCM under a nitrogen atmosphere followed by the addition of DIPEA (0.045 g, 0.35 mmol) and 6-azidohexanol (3) (0.05 g, 0.35 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with water and the product was extracted with DCM. The organic layer was separated and dried over sodium sulfate (Na2SO4), concentrated under vacuum to get the crude derivative 4. The solid was then washed with acetonitrile to remove excess HBTU and precipitates were filtered out. The resulting product was re-dissolved in dry DCM and cooled at 0oC. TFA (2.5 ml) was added dropwise and mixture was allowed to stir at room temperature for 2 hours. After completion of the reaction, DCM was removed and the crude was purified by column chromatography using pure EtOAc as eluent to yield the intermediate, Pro-Cip, in 68% yield. 1H NMR (DMSO-d6, 300 MHz) L 8.55 (s, 1H), 8.07 (d, J = 13.5 Hz, 1H), 7.31 (d, J = 4.8 Hz, 1H), 4.35 (t, J = 6.6 Hz, 2H), 3.98-



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387 (m, 1H), 3.49-3.43 (m, 1H), 3.33-3.29 (m, 6H), 3.18-3.15 (m, 4H), 1.88-1.79 (m, 2H), 1.711.62 (m, 2H), 1.55-1.45 (m, 4H), 1.39-1.33 (m, 2H), 1.19-1.14 (m, 2H); 13C NMR (DMSO-d6, 75 MHz) L 173.22, 165.47, 155.02, 151.73, 148.08, 144.84, 144.70, 138.02, 122.87, 113.17, 112.87, 109.93, 104.98, 64.61, 51.35, 50.35, 45.54, 34.63, 28.74, 28.62, 26.40, 25.60, 8.14; MS (ESI): m/z calcd. for C23H29FN6O3: 456.2285 Found: 457.2352 [M+1]+. 2.3. Characterization of Small Molecules H-Cy and Pro-Cip The chemical structures of the synthesized small molecules were confirmed by Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR), 1H and 13C nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS). The 1H- and

13C-NMR

spectra were

recorded on Bruker 300 NMR instruments. Chemical shifts are reported as L values (ppm) with (residual) solvent as an internal standard (DMSO-d6; 1H: L = 2.50, 13C: L = 39.52 and CDCl3; 1H: L = 7.26, 13C: L = 77.16). Column chromatography purifications were performed by using silica gel 60 (70 ~ 230 mesh) as a stationary phase. Analytical thin-layer chromatography (TLC) was conducted using TLC silica gel 60, visualized under ultraviolet light. All UV-Vis spectral studies were carried out on Ultrospec 4300 pro spectrophotometer. 2.4. Fabrication and Characterization of Membranes All the membranes including membranes M-H-Cy and M-Pro-Cip (incorporating chromogenic probe H-Cy and Pro-Cip, respectively), pure PU membrane, and theranostic wound dressing (THWD) were fabricated via the blend electrospinning technique (Figure S1, Supporting information). Depending upon the type of membranes, the small molecules and PU were dissolved in 1:1 DMF:THF mixture according to composition shown in Table S1 (Supporting information). The mixture was stirred overnight at 40 °C so that a clear viscous solution was formed. The solution 8 ACS Paragon Plus Environment

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was then subjected to electrospinning using an NE300 electrospinning apparatus (Inovenso, Turkey), with the parameters set at a flow rate of 0.25 mL/h and voltage of 12 kV. The distance of the collector from the tip of the spinneret was maintained at 15 cm. During electrospinning, the fibrous electrospun membranes were collected on an aluminum foil adhered to the collector. The fibers were dried in a vacuum oven at room temperature for 24 hours. The electrospun membranes were characterized by scanning electron microscopy (SEM, Instrument model no. Quanta 650 FEG ESEM). Prior to analysis, samples were sputter coated with gold-palladium. The mean fiber diameters were calculated based on the SEM images. 2.5. Chromogenic Response and Antibacterial Activity of Theranostic Membrane (TH-WD) The colorimetric response of the membrane was observed by naked eye. Briefly, the membrane was cut into small squares with dimension of 1 cm × 1 cm and put into glass vials followed by the addition of 1 ml of bacteria culture (108 CFU/ml) of Pseudomonas aeruginosa (ATCC 27853). For the control, the bacterial suspension was replaced by pure LB broth solution. Both the vials were incubated for predetermined time durations at 37 °C. The membranes were then taken out of the vials and observed for color change. For the antibacterial properties of TH-WD, the standard plate counting method was followed. An aliquot of 30 µl bacterial solution was taken from the vial containing TH-WD and subjected to serial dilutions (10 fold each) using 0.1 M PBS. 30 µl of each dilution was then spread onto a nutrient agar plate. After 18 hours incubation at 37 °C, the viable bacteria colonies were counted on the agar plates. Each experiment was conducted in triplicate. 2.6. Cytotoxicity Assay The cell cytotoxicity of the membranes towards the skin fibroblast cells (ATCC® PCS-201) was evaluated by WST-1 assay kit using an indirect contact method.



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2.6.1. Indirect Contact Method In this method, there is no direct interaction between the cells and material used (Figure S2, Supporting information). Instead, it involves the interactions between leachable molecules from the material with the cell monolayer. The material is dropped into a transwell insert (Corning, Corning, NY) which in turn is placed into an outer well seeded with cells and culture media just above the cell surface, ensuring that the insert is submerged into the cell media. For all assays, cells cultured under normal condition (i.e., PBS) without any membrane materials were used as blank controls. PU-membrane and TH-WD were used as a negative control and a test sample, respectively while H2O2 was used as a positive control. 2.6.2. Cell Culture and WST-1 Assay The fibroblast cells (ATCC® PCS-201) were cultured in a T75 cell culture flask using basal media supplemented with 2% fetal bovine serum, and 7.5 mM L-Glutamine (GibcoTM) under 37 oC, 5% CO2 and 95% relative humidity. Each day, the culture media was removed and replaced with freshly prepared media. When the cells reached 70-80% confluence, they were detached with 0.25% trypsin and dispersed in 10 ml of basal media, achieving a cell density of 2 × 105 cells/ml. Then, 0.5 ml of above dispersed medium was transferred to each well in a 24 multi-well plate and allowed to stand overnight so that cells adhere to the surface. Meanwhile, the electrospun membranes (TH-WD and pure PU membranes) were cut into four round pieces with 4 mm diameter. These membrane pieces were disinfected by immersing them into 70% EtOH for 10 minutes. After being dried, each membrane piece was dropped into the transwell inserts (in quadrant replicates) and placed in the 24 multi-well-plate. After 24 hours incubation, the inserts were removed and the culture media in the outer well was replaced with fresh media containing 10% WST-1 reagent. Plates were incubated for another 1 hour at 37 °C. Then, 100 µL aliquots


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from each well (in quadrant replicates) were transferred to 96-well plates and viability of cells was evaluated using spectrophotometer at 570 nm wavelength. 2.7. Ex Vivo Detection of Bacterial Infection Uninjured samples of pig skin were cut into small portions with a dimension of 3.0 cm × 3.0 cm. To create wounds on the skin, a brass rod with dimension of 2 cm × 2 cm was heated in boiling water for 10-15 minutes and then subsequently placed on the center of each skin sample under its own weight (9.2 N) for 1 minute. The skin samples were then immersed into saline solution (0.9% NaCl) for 15 minutes and then disinfected with 70% EtOH. The samples were transferred into sterile Petri dishes and allowed to dry in the open. An aliquot of 200 ZE from an overnight culture of P. aeruginosa (108 CFU/ml) was deposited on the wound and spread to cover the wound area using a glass spreader. Subsequently, TH-WD was used to cover the infected wound and images were recorded after 2 and 4 hours incubation at 37 °C in a moist environment. In the control experiment, TH-WD was placed on a similar wound that only contained LB broth. To quantify the bacterial count on the wound bed, a small portion of skin was punched from the wound bed after different times of incubation using a 0.4 mm Integra™ Miltex™ Standard Biopsy Puncher (0.1256 cm2). Each skin portion was then suspended into 1 ml PBS in a vial and sonicated for 2 minutes to detach the bacteria from the skin surface followed by 20 seconds of vortexing in order to uniformly disperse the bacteria. An aliquot of 30 µl was taken from the vial and subjected to serial dilution using 0.1 M PBS. The remaining procedure for the antibacterial studies has been outlined under section 2.5.



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3. RESULTS AND DISCUSSION The hemi-cyanine based chromogenic probe (H-Cy) and prodrug of ciprofloxacin (Pro-Cip) were synthesized as shown in Scheme 1. Briefly, probe Cy was synthesized by EDC (1-ethyl-3-(3dimethylaminopropyl) carbodiimide) coupling of hemi-cyanine derivative 2 with 6-azidohexanoic acid while prodrug Pro-Cip was synthesized by HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate) coupling of Boc-protected ciprofloxacin derivative 3 with 6-azidohexanol followed by Boc de-protection in the presence of trifluoroacetic acid. All the intermediates and products were well characterized by 1H/13C NMR, MALDI-TOF and ESI-MS. (Figure S8-S16, Supporting information).

NC

CN

NC

HO

CN

+

EDC, DMAP

N3

HOOC

O

O

CN O

NC

4

N3

O

DCM, r.t.

2

H-Cy

1

O F

O

O F

OH HO

N

N

N

O O

N

N

F

N3

4 THF, TFA

N

O O

4

N3

N

HN

N

O O

Boc-Cip

O

N3

3 HBTU, DIPEA

O

O

4

Pro-Cip

Scheme 1. Synthetic scheme for the synthesis of H-Cy and Pro-Cip First, we investigated the lipase-responsive spectroscopic behavior of chromogenic probe H-Cy in the presence of purified lipase using UV-Vis spectroscopy. For this, we employed lipase from Candida rugose since it is the most frequently used lipase-producing yeast, easily available and economically affordable. Moreover, the three distinct forms of extracellular lipases (Lip A, Lip B and Lip C)24,25,26 isolated from Candida rugose have also been reported to be produced by P. aeruginosa.27,28 Spectroscopically, H-Cy exhibits a sharp absorption maximum at 424 nm in 5% DMSO in phosphate buffered saline (PBS, pH 7.4, 37 oC). Upon incubation of H-Cy (50 µM) with purified lipase (10 µg/ml) in 5% DMSO/PBS at 37 oC, the enzyme-mediated ester hydrolysis alters 12 ACS Paragon Plus Environment

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the dye, as revealed by a gradual decrease of the absorbance band at 424 nm with concomitant increase of a red-shifted new band at 575 nm (Figure 1a) which is characteristic of hemi-cyanine dyes. Each reading was recorded at intervals of 5 minutes for a total of 55 minutes. Moreover, these changes were accompanied by a solution color change from yellow to red (Figure 1b). 1.2

1.2

(a)

(b)

1.0

1.0

0.8

0.8

Absorbance

Absorbance

(-) lipase

0.6 0.4 0.2

(+) lipase (-) (+)

0.6 0.4 0.2

0.0

0.0 300

400

500

600

700

300

400

Wavelength (nm)

500

600

700

Wavelength (nm) 1.2

0.9

(c)

(d) 1.0

Absorbance at 424 nm

0.8

Absorbance at 575 nm

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


0.7 0.6 0.5 0.4 0.3 0.2

0.8 0.6 0.4 0.2

0.1 0.0

0

10

20

30

40

Time in minutes

50

60

0

2

4

6

8

10

12

Time in hours

Figure 1. (a) The absorption spectra of H-Cy (50 µM) in the presence of lipase (10 µg/ml) recorded after each 5 minutes for 55 minutes. (b) The absorption spectra before (black) and after (red) addition of lipase. Inset shows the color change of solution. (c) Plot of absorbance of H-Cy at 575 nm versus time in minutes. (d) Plot showing the photo-stability of probe H-Cy in the absence of lipase with time. All spectra were recorded in PBS (0.01 M, pH = 7.4) containing 5% DMSO. These spectral changes were attributed to the fact that in the absence of lipase, there is negligible or no intramolecular charge transfer (ICT) from the phenol moiety to the cyanine moiety in H-Cy since the hydroxyl group is engaged in ester bond formation. However, after hydrolysis of the ester linkage by lipase, ICT is enhanced from the phenolate ion to the cyanine moiety, which is responsible for the color change. As shown in Figure 1c, an excellent linear correlation (R2 = 0.996) between the time of incubation and absorbance was obtained under physiological 13 ACS Paragon Plus Environment


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2238 cm-1. The appearance of the broad beak (represented by a black dotted circle) at 2300 cm-1 may be indicative of the click reaction. Unfortunately, the chromogenic response of the clickfunctionalized membrane in the presence of purified lipase was not fast enough. It took about 2 days of incubation (at 37 oC) to show color change. This slow response may be attributable to the low surface density of clicked H-Cy. (a)

1000

2000

3000

4000

(b)

O

0.297

0.198

1000

O PSI

2000

3000

4000

H-Cy clicked membrane

0.36

N

0.24 x

(c)

0.12 0.00

0.099

Transmittance

Transmittance

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


0.000 O

O

0.198

N H O

N

0.132

O

x

HN

y

PU+PSI-PA membrane

0.182 0.091 0.000 PU membrane

0.18 0.12

PSI-PA

0.066

0.273

0.06 0.000

0.00 1000

2000

3000

Wavenumber (cm-1)

4000

1000

2000

3000

4000

Wavenumber (cm-1)

Figure 2. (a) The FTIR spectra of polysuccinimide (PSI), and propargyl functionalized polysuccinimide (PSI-PA), (b) The FT-IR spectra of membranes fabricated from: pure polyurethane (PU), bend mixture of PSI-PA and PU, and surface modification with H-Cy. (c) Images of membrane before (upper row) and after (lower row) surface modification with H-Cy. We therefore employed an alternate strategy in which small molecules were directly blended with PU prior to electrospinning. This technique has its own advantages. Firstly, the blend electrospinning technique enables the manipulation of the sensitivity of biomaterial by altering the loading concentration of the responsive small molecule conjugate (H-Cy). Secondly, it allows the uniform distribution of small molecules throughout the membrane surface. By using this strategy,




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(a)

(b)

(c) Bacterial reduction (% CFU/mL)

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

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(d) 100

P. aeruginosa

80

60

M-Pro-cip 40

O F

20

N

O

O OR

1

2

Time in hours

4

N

N

HN

0

F

P. aeruginosa

O OH

N

HN

Prodrug (Pro-Cip) R = alkyl chain

Ciprofloxacin (active drug)

Figure 5. The chromogenic response of M-H-Cy membrane at different concentrations of lipase upon incubation at 37 oC for 1 h (a; from left to right; pure PBS, 0.004, 0.025, 0.1 and 0.5 mg/ml) and overnight (b; from left to right; pure PBS and 0.5 mg/ml). (c) The antibacterial properties of M-Pro-Cip membrane towards P. aeruginosa (ATCC 27853, 1.62 × 108 CFU/ml) at different time intervals. (d) Schematic representation of drug activation by P. aeruginosa. After confirming the chromogenic response of M-H-Cy membrane and the antibacterial property of M-Pro-Cip membrane, we then added both small molecules into 1:1 DMF:THF solution of PU and electrospun the mixture into a nanofibrous membrane, termed as theranostic wound dressing (TH-WD). SEM images (Figure S3-S4, Supporting information) showed a smooth bead-free network-like morphology with an average nanofiber diameter of 480 nm. The theranostic properties of TH-WD were investigated against P. aeruginosa (ATCC 27853). As shown in Figure 6a (orange bars), TH-WD also displayed efficient inactivation of P. aeruginosa.


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(a) Bacterial reduction (% CFU/mL)

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(b)

100

P. aeruginosa

P. aeruginosa

E. coli

-ve

+ve

75

E. coli

50

-ve

+ve

25

0 1

2

4

Time in hours

(c)

Bacterial Infection

TH-WD

Ciprofloxacin

H-Cy (active form)

Figure 6. (a) The antibacterial properties of theranostic wound dressing (TH-WD) towards P. aeruginosa (ATCC 27853) and E. coli (TOP 10) at different time intervals, (b) The color response of TH-WD membrane in the presence of P. aeruginosa and E. coli (TOP 10) after 4 hours incubation at 37 oC. (c) Schematic representation of the activation of H-Cy dye and prodrug ProCip under bacterial infection. The antibacterial efficiency of TH-WD increased with increase in incubation time and 100±4% reduction of P. aeruginosa (ATCC 27853) was achieved after 4 hours of contact. Interestingly, bacterial reduction was accompanied by a membrane color change from yellow to green similarly as was observed when incubating the M-H-Cy membrane in the presence of purified lipase. Bacterial lipase is an extracellular enzyme excreted by bacterial cells during the growth phase.30 The bacterial suspension used in the antibacterial test was pre-incubated for 18 hours at 37 oC to reach the target concentration (108 CFU/mL). There may exist a sufficient amount of extracellular



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lipase in the bacterial suspension that can effectively and simultaneously catalyze the hydrolysis of both small molecules (H-Cy and Pro-Cip). It is observed that even after 100% bacterial reduction, lipase activity is still present - which is observed as the concomitant color change of the membrane. Once pro-Cip has been converted to active Ciprofloxacin, the compound can effectively reduce the viability of P. aeruginosa by 100%. In turn, P. aeruginosa can no longer produce and secrete any more lipase. Therefore, Ciprofloxacin inhibits the production of new lipase in the bacterial suspension. However, Ciprofloxacin does not have an effect on the lipase that was produced beforehand. In this case, any lipase that was produced before the initiation of pro-Cip to Ciprofloxacin may still remain active and continue to act on available substrates such as H-Cy. This explains the continued color change expression even after the population of P. aeruginosa has been reported to be reduced by 100%. Similar chromogenic response was also observed with other lipase producing bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa PA01 (Figure S5, Supporting information). The chromogenic and antibacterial properties of TH-WD may be attributed to the diffusion of ProCip and H-Cy from inside the membrane to the incubation solution followed by activation by extracellular lipase. The diffusion of Pro-Cip and H-Cy was supported by the recorded timedependent UV-Vis spectra. TH-WD was immersed in water without lipase addition. Interestingly, after 30 minutes of incubation, UV-Vis spectra of the water layer showed two strong bands at 272 nm and 438 nm which increase in intensity with increasing incubation time. These bands, respectively, correspond to pro-Cip and H-Cy. However, no such bands were observed for the water fractions taken immediately after immersing the TH-WD. From these results, we speculate that upon immersing TH-WD in solution, the PU-scaffold became swollen by absorbing water, 20 ACS Paragon Plus Environment

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about 35% of its weight. The swelling property of the PU has already been well established in the literature.31 It is speculated that this allowed the small molecules, Pro-Cip and H-Cy, to diffuse out from the membrane matrix to the solution. The release of both Ciprofloxacin and the active dye from TH-WD in response to lipase was confirmed by UV-Vis and ESI-MS. A piece of TH-WD membrane with the dimension of 1 cm × 1 cm was immersed in lipase solutions at different concentrations and incubated at 37 oC for 1 hours. The UV-Vis spectra of solutions were then recorded which exhibited two sharp bands at 275 nm and 575 nm (Figure S7, Supporting information). These bands are characteristic of Ciprofloxacin32 and the active dye,33 respectively. Also, the intensity of these bands increased with increasing concentration of lipase or incubation time. Moreover, during this time the color of the solution changed from colorless to red which can be attributed to the active dye released in the solution. The ESI-MS analysis of solution showed two strong peaks at (m/z) 332.14 (Mcip+1) and 304.11 (Mdye+1), corresponding to Ciprofloxacin and the active dye, respectively (Figure S16, Supporting information). Further, to validate the bacterial lipase-responsiveness of membrane, the theranostic studies were performed in the presence of E. coli Top10, a low lipase-secreting bacterium.34 TH-WD showed neither color change (Figure 6b) nor any significant antibacterial property (6a, black bars) in the presence of E. coli (TOP 10). These results clearly suggested that membrane is selectively activated by lipase-secreting bacteria, thereby facilitating an on-demand release of drug/dye at the infection site (Figure 6c). In addition, since the biocompatibility of biomaterials is a major concern for biomedical applications, WST-1 assay was conducted to check the cytotoxicity of TH-WD. Figure 7 presents the WST-1 assay results of fibroblast cells (ATCC® PCS-201) after 24 hours incubation with different treatments. 21 ACS Paragon Plus Environment


120

40

Control

100

Cell viability (%)

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

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-ve control (only PU membrane) 80

Experimental (Theranostic membrane)

60 40

0.7 mM H2O2 (+ve Control) 20

1.0 mM H2O2 (+ve Control)

0 1

2

3

4

5

Treatments

Figure 7. WST-1 assay showing the comparison of cell viability using five different treatments against the fibroblast cells. (1= Control, 2 = PU-membrane, 3 = TH-WD, 4 = 0.7 mM H2O2, and 5 = 1.0 mM H2O2. The data are presented as mean ± S.D (n=4).

As a positive control, hydrogen peroxide (H2O2) caused reduction in cell viability to 52±2% at a concentration of 0.7 mM and to 5.0±0.3% at 1.0 mM. However, TH-WD and PU membrane did not show any cytotoxicity against fibroblast. These results suggested that both TH-WD and PU membrane are biocompatible with the cells. Finally, we performed an ex vivo study with a pig skin model of full-thickness burn to demonstrate the ability of TH-WD in colorimetric detection and treatment of bacterial infection. A burn wound on the pig skin was inoculated with P. aeruginosa at a concentration of 3.2 × 108 CFU/cm2 followed by placing TH-WD on the wound. Color change was inspected visually after 4 hours incubation at 37 oC in a moist environment (Figure 8A). Interestingly, no color change was observed for TH-WD placed on normal wound (i.e., un-effected), indicating that TH-WD responds specifically to the wound infection.


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27853). These changes are attributed, respectively, to the release of the antibiotic (Ciprofloxacin) and the active dye, hemi-cyanine, respectively, via lipase-mediated hydrolysis of the ester linkages in the prodrug and the chromogenic probe. Since drug release is achieved only in the presence of pathogenic bacteria, the apparent concentration of drug can be controlled which ultimately reduces the risk of bacterial resistance that is associated with the overuse of antibiotics. Other reported bacteria-sensitive dressings only detect mature bacterial biofilms in the wound, which by then, is already too late for timely and effective treatment and not clinically meaningful. In contrast, our novel dressing presents clear color change from yellow to dark green as well as efficient antibacterial properties in an ex vivo study within 4 hours after being placed on a freshly inoculated wound on pig skin. These qualities were accomplished with minimal cytotoxicity, providing a sensitive, effective and safe theranostic wound dressing. ASSOCIATED CONTENT Supporting Information The characterization data such as 1H and 13C NMR and mass spectra, SEM images for TH-WD and the schematic representation of blend electrospinning for membrane fabrication are given in Supporting Information. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Fax: (204) 474-7512 ORCID iDs Hardev Singh: 0000-0001-7236-8662 Mohammad Reza Kazemian: 0000-0001-7753-9651 24 ACS Paragon Plus Environment

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Runqiang Yang: 0000-0002-0191-6315 Chengbo Yang: 0000-0003-4449-5132 Song Liu: 0000-0003-0301-9535 ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Research Manitoba Mid-Career Operating Grant, the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (Grant no. RGPIN/04922-2014), and the Collaborative Health Research Projects (CHRP) operating grant (Grant no: CHRP 413713-2012). The authors also gratefully acknowledge the technical assistance of Alireza Kianrokh in creating the cover art.

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Graphical Abstract

diagnosis unit (dye) inactive drug

Skin

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Page 30 of 30

dead bacteria

bacterial enzyme

converted dye active drug

live bacteria Infected wound bed


Lipase-Responsive Electrospun Theranostic Wound Dressing for

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