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Construction of high drug loading and enzymatic degradable multilayer films for self-defense drug release and long-term biofilm inhibition Bailiang Wang, Huihua Liu, lin sun, yingying jin, xiaoxu ding, lingli li, jian ji, and Hao Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01268 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 3, 2017

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Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Construction of high drug loading and enzymatic degradable multilayer films for self-defense drug release and long-term biofilm inhibition Bailiang Wanga, b*, Huihua Liub, Lin Suna, Yingying Jina, Xiaoxu Dinga, Lingli Lia, Jian Jic,*, and Hao Chena, b* a

School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University,

Wenzhou, 325027, China b

Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences,

Wenzhou, 32500, China c

MOE Key Laboratory of Macromolecule Synthesis and Functionalization,

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China * Corresponding author. Fax: +86 577 88017524. E-mail: [email protected] (B.L. Wang); [email protected](J. Ji); [email protected] (H. Chen) Abstract: Bacterial infections and biofilm formation on the surface of implants is an important issue which greatly affect biomedical applications and even cause device failure. Construction of high drug loading systems on the surface and control the drug release on-demand is an efficient way to lower the development of resistant bacteria and biofilm

formation.

In

the

present

study,

ACS Paragon Plus Environment

(montmorillonite/hyaluronic

Biomacromolecules 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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acid-gentamicin)10 ((MMT/HA-GS)10) organic/inorganic hybrid multilayer films were alternately self-assembled on substrates. The loading dosage of GS was as high as 0.85 mg/cm2 which could be due the high specific surface area of MMT. The obtained multilayer film with high roughness gradually degraded in hyaluronidase (HAS) solutions or bacterial infection microenvironment which caused the responsive release of GS. The release of GS showed dual enzyme and bacterial infections responsiveness which also indicated good drug retention and on-demand self-defense release properties of the multilayer films. Moreover, the GS release responsiveness to E. coli showed higher sensitivity than that to S. aureus. There was only ~5 wt% GS release from the film in PBS after 48h immersing and the amount quickly increased to 30 wt% in 105 CFU/mL E. coli. Importantly, the high drug dosage, smart drug release and the film peeling from the surface contributed to the efficient antibacterial properties and long-term biofilm inhibition functions. Both in vitro and in vivo antibacterial tests indicated the efficient sterilization function and good mammalian cells and tissue compatibility. Keywords:

Controlled

release;

antibacterial;

microenvironment;

enzymatic

degradation; multilayer films

1. Introduction

Bacteria can easily colonize on the surface of implants during and after surgical operation due to patients having compromised immune systems

1, 2

. As it is known,

more than 50% of the hospital acquired infections are associated with the adherence


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of bacteria on implants and other biomaterials3, 4. Till now, the routine way to deal with implants related infections is intravenous injection of antibiotics in a few days after implantation5, 6. In addition, the abuse of antibiotics in other areas lead to the generation of resistant bacteria and the situation is growing more and more serious. What's worse is the emergence of super resistant bacteria which is resist to almost all of the antibiotics7, 8. Especially in Intensive Care Unit (ICU), the ubiquitous presence of super resistant bacteria directly threatens the patient's life. As a result, construction of antibacterial coatings on implants surface has been paid great efforts to lower the bacterial infections rate9, 10. Among them, local delivery of high dose of antibiotics around the implants plays an important role in prevention and treatment of bacterial infections11, 12. However, excessive release and release below the minimum inhibitory concentration also lead to the development of resistant bacteria. Therefore, design and preparation of multilayer films on implants to load high dosage of small molecule antibiotics and controlled release of the drug in an on-demand self-defense manner is of great advantages13-15. Many kinds of drug delivery systems have been constructed including nano/micron carriers, hydrogels, coatings and bulk materials etc. to load drugs and control the release behavior of the drug from the systems16-19. The first of the drug delivery systems studies, drug release was tuned to a sustained way through physical embedding or electrostatic forces with the matrices. In recent years, advanced drug delivery systems are emerging to combine specific stimuli, such as pH value, temperature, electric and magnetic fields, redox and light responsive, into the matrices



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or through crosslinking to tune the permeability of the matrices and eventually control the drug release from the systems17, 20-23. For instance, S. Sukhishvili et al. prepared tannic acid/cationic antibiotics (gentamicin, tobramycin or polymyxin B) multilayer film via self-assembly method13, which showed bacterial responsive drug release as the pH value decreased. Hammond et al. firstly synthesized hydrolysable poly (β-amino

ester)

and

constructed

(Poly

(β-amino

ester)/hyaluronic

acid)1

(gentamicin/hyaluronic acid)1) multilayer films ((PAE/HA)1(gentamicin/HA)1)24. The degradation rate of the films, loading dosage and release behavior of the drug could be adjusted through changing chain length of the PAE. It was also demonstrated in our previous studies that thermal crosslinking or bionic dopamine cross-linking of the drug loaded polyacrylic acid/polyethyleneimine multilayer films reduced the release rate of gentamicin sulfate (GS)25. However, the drug loading dosage was only 160 µg/cm2 and more than 80 % of the drug released in a few days after immersing in PBS. How to load high dosage of antibiotics and endow the systems with self-defense film degradation and drug release performances is an important and urgent issue. Layer-by-layer (LBL) self-assembly method has been widely used in biomaterials surface modification and advanced drug delivery systems21,

26, 27

. There are some

significant advantages for this technology including simple construction process, independent of substrate and precise controllability of the thickness and so on. Furthermore, stimulus responsive factors can easily be combined into the multilayer films and antibacterial agents can be triggered release from the drug delivery systems28-30.

S.

Sukhishvili

et

al.

developed


pH/bacteria-responsive

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(montmorillonite/polyacrylic acid)n ((MMT/PAA)n) multilayer films to host small-molecule antibiotics which showed multiple mechanisms of antibacterial functions including the permanent retention of antimicrobials, bacteria-triggered release of antibiotics and bacteria-induced film swelling31. MMT as a kind of nanoplatelets, can be included within LBL films to load a high percentage of drugs and block its release due to its high specific surface area 32. Once bacterial infections happen on the implants, the microenvironment greatly changed such as pH reduction, specific enzyme concentration increase and virulence factor release et al.13,

33, 34

.

Hyaluronidase (HAS) and chymotrypsin obviously increase in the bacterial infections microenvironment which can be used to trigger drug release from the matrices through enzymatic degradation35, 36. It is noteworthy that enzyme response with high efficiency, sensitivity and specificity is superior to other stimulus response factors. In this work, we aimed to combine MMT with high drug loading dosage and retention properties and HA responsive to HAS on biomaterials surface to endow the multilayer film with on-demand self-defense drug release property for long-term biofilm inhibition. In recent years, self-defense antibacterial surfaces especially drug delivery systems that are responsive to bacterial infection microenvironment have drawn more and more attentions37-39. The on-demand self-defense strategy reduces the ineffective release of drugs and reduces the risk of developing of antibiotic-resistant bacterial strains. It has been proven in our previous study that through compounding pH responsive antibiotics-loaded micelles into multilayer films, the drug release was



responsive to local acidic bacterial infections microenvironment34. The drug delivery systems showed both bacteria-triggered and pH-responsive release properties and can be used as self-defensive antibacterial coatings. GS as one of the most commonly used antibiotics has been loaded and demonstrated high efficacy antimicrobial properties against a broad spectrum of bacteria that are known to cause infections associated with biomedical implants, such as E. coli, S. epidermidis and S. aureus40, 41. Furthermore, controllable and gradually film peeling from the surface through film degradation play a critical role on bacteria anti-adhesive and long-term biofilm inhibition42-44. As indicated in our previous work, controlling composite multilayer films hydrolysis through thermal crosslinking, it showed top-down degradation process and led to almost no adhesion of bacteria in 24 h45. It was demonstrated in other report that enzyme degradation of HA led to the fully inhibition the growth of bacteria after 24 h of incubation with the (HA-cateslytin /chitosan)n multilayer films46.

Figure 1. Schematic representation of the (MMT/HA-GS)n self-assembled multilayer films and the enzyme-triggered and bacteria-triggered release of GS.

More recently, bacteria-triggered and on-demand drug release systems have attracted great attention because of the highly efficient and specifically targeted


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properties. In this work, as illustrated in Figure 1, (MMT/HA-GS)n organic-inorganic hybrid multilayer films were developed to load GS antibiotics. GS was embedded in the organic-inorganic hybrid multilayer films in the deposition process for a high loading dosage. With positive charges, GS can combine with both MMT and HA based on electrostatic interactions. Based on the high specific surface area and lamellar structure, MMT not only combined with small GS, but also served as barriers to block GS release from the films. HA in this system could be progressively enzymatic degraded in the presence of HAS or bacterial infections which led to drug release and film peeling. The peeling of film components from the surface can resist bacteria adhesion and long-term biofilm inhibition. Spectroscopic ellipsometry and SEM tests were used to determine the thickness of the multilayer films before and after films enzymatic degradation. The loading dosage and drug release behavior were measured by spectroscopy method. The effects of HAS concentration, bacteria species and concentration on films degradation and GS release rates were investigated. The bactericidal and long-term biofilm inhibition properties of the multilayer films were investigated in detail both in vitro and in vivo.

2. Materials and methods

2.1. Materials and reagents

Polydimethylsiloxane (PDMS) was purchased from Dow Corning (Sylgard®184) and made into solid according to the instructions, using 10:1 ratio of elastomer base to curing agent. HA was purchased from Freda Biochem Co., Ltd. Branched



polyethyleneimine (PEI, Mw: 25 kDa), hyaluronidase (HAS, from bovine testes, Type I-S, lyophilized powder, 400-1000 units/mg solid) and GS were purchased from Sigma-Aldrich. A Millipore MilliQ system (USA) was used to produce Ultrapure distilled water.

2.2. Construction of the (MMT/HA-GS)10 multilayer films

Substrates such as glass discs, PDMS and silicon wafers were successively cleaned in ethanol, acetone and water before using. The MMT stock solution (5 mg/mL) was prepared 2 weeks before using. The MMT deposition solutions were prepared by diluting the stock MMT solution into 0.5mg/mL (pH=2.5) and were dispersed by ultrasonic treatment overnight. HA and GS in water were dissolved at 1 mg/mL and 0.5 mg/mL respectively (pH 2.5) for (MMT/HA-GS)10 multilayer films deposition. Substrates were deposited firstly in PEI solution (5 mg/mL, 30 min) for a precursor. To construct the (MMT/HA-GS)10 multilayer films, the substrates were alternately dipped in HA-GS solution and MMT solution for 10 min and washed in the buffer solution (pH=2.5) until the numbers of bilayers were obtained. The (MMT/HA-GS)10 hybrid multilayer films were dried under a gentle stream of N2. This dipping cycle corresponds to the deposition of one bilayer.

2.3. Multilayer films thickness measurement

In the process of multilayer films preparation, the changes of thickness were tracking


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tested by spectroscopic ellipsometry (M-2000 DITM, J.A. Woollam). This measurement was carried out on silicon wafers. The continuous wave length ranging from 124 to 1700 nm and angle of incidence of both 65º and 70º were chosen for the ellipsometry measurements. ∆ and Ψ values measured at wavelength of 600-1700 nm were chosen for data analysis. Cauchy model was applied for determining the thickness of multilayer films. Parameters of An and Bn for Cauchy layer were set as 1.45 and 0.01 respectively as fit parameters. Then the thickness that best fit the multilayer films could be automatically calculated.

2.4. Enzyme and bacterial responsive degradation of the multilayer films

The obtained multilayer films were immerged in HAS (88 or 175 U/mL) solutions or bacteria (S. aureus or E. coli with the concentration at 103 or 105 CFU/mL) in 0.01 M PBS for 48 h. The samples were taken out at certain time interval and dried by N2 for thickness measurement by spectroscopic ellipsometry and SEM (SiRion100) cross section observation. The surface morphology changes of the multilayer films on silicon wafers were also examined by SEM after drying.

2.5. Release of GS from (MMT/HA-GS)10 multilayer films

The total GS loading dosage in the multilayer films was calculated through deducting the remaining GS in HA-GS solution and two buffer solutions from input GS amount. For the release process, GS-loaded multilayer films were introduced into a 5 mL of PBS buffer solution (pH=7.4) for 48 h. For each point, 300 µL of the PBS buffer



solutions were sampled and the release of GS was measured. Then, solutions were reintroduced in the plate for other points and the release kinetics profiles were obtained. GS concentrations were measured using a procedure described by Sampath and Robinson 47. Briefly, the o-phthaldialdehyde reagent was formulated by adding 2.5 g o-phthaldialdehyde, 62.5 ml methanol and 3 ml 2-mercaptoethanol to 560 ml sodium borate solution, pH 8. The reagent was stored in a brown bottle in a dark chamber for at least 24 h before use, as it is light sensitive. The GS aliquot, o-phthaldialdehyde reagent, and isopropanol were mixed in equal proportions and stored for 30 min at room temperature. The ophthaldialdehyde reacted with the GS amino groups and chromophoric products were obtained, the absorbances of which were measured at 332 nm using a Genesys Spectronic 20 spectrophotometer (Spectronic Instruments, Rochester, NY). A calibration curve was used to calculate the GS concentrations in the samples.

2.6. In vitro antibacterial test

Antimicrobial tests of (MMT/HA-GS)10 multilayer films were conducted qualitatively and quantitatively by bacterial LIVE/DEAD staining method respectively with E. coli (ATCC 8739) and S. aureus (ATCC 6538) as model bacteria. A LIVE/DEAD BacLight bacterial viability kit (L-7012, Invitrogen) was used to determine bacterial cell viability. This test evaluates the structural integrity of bacterial membrane. The (MMT/HA-GS)10 multilayer films modified and control PDMS sheets were incubated


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with S. aureus or E. coli for 24 h and stained according to the kit protocol. After careful washing, the samples were sealed with tin foil and observed by fluorescence microscope investigation (Zeiss, Germany). Zone inhibition test was carried out with a modified agar diffusion assay. The GS-loaded multilayer films were immersed in 0.1 M PBS for 24 h, 3 d, 5 d, 8 d and 20 d, respectively for the long-term antibacterial property measurement. Then the samples were placed on nutrient agar in Petri dishes which had been seeded with 0.2 mL 1.0 × 106 cells/mL of S. aureus or E. coli bacteria suspension. The Petri dishes were examined for zone of inhibition after 24 h incubation at 37 ºC. The area clearing surrounded the film where bacteria were not capable of growing was reported as the zone of inhibition. To test the bactericidal property, the multilayer films was examined through contacting with bacteria solutions and measured by flat counting method. The (MMT/HA-GS)10 multilayer films modified and control PDMS sheets were incubated with S. aureus or E. coli (1.1 × 105 CFU/mL) for 1 h at 37 °C. Then the bacterial solution (0.2 mL) before (0 h) and after contacting (1 h) solution was spread onto triplicate solid agar, and the number of viable bacteria was counted after 24 h incubation.

2.7. Animal experiments of antibacterial activity

The study was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Experiments were carried out in New Zealand white rabbits, weighing between 2.5 and 3.5 kg (obtained from the Animal Administration



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Center of Wenzhou Medical University). The rabbits were treated in accordance with guidelines set forth by the Association for Research in Vision and Ophthalmology. Two groups of animals were studied for native PDMS and (MMT/HA-GS)10 multilayer films modified PDMS respectively. All implants were sterilized prior to implantation and stored in sealed Petri dishes. The animal model used conformed to that described in our previous work16. New Zealand White rabbits were anesthetized using isoflorane delivered by facemask. A broad area of the back was shaved, and the underlying skin washed with surgical scrub, wiped with alcohol, painted with betadine, and draped for surgery. Under sterile conditions, two subcutaneous symmetrical multilayer films-coated and uncoated PDMS implants were created on either side of the spine; care was taken to ensure

broad

physical

separation

between

the

implants

to

eliminate

cross-contamination risks. In the rats, the subcutaneous pocket was then inoculated with 0.2 mL of the 108 cells/mL S. aureus suspension. The incisions were closed in a single layer with 4-0 interrupted nylon suture and the epidermis was cleaned with 2 % H2O2. Rats were housed individually and given ad libitum access to food and water. The animals were then observed daily throughout a 1 week period, after which they were returned to the operating room and prepared as above. Each pocket was opened via a small incision distinct from the prior wound, through which two sterile cotton swabs were inserted, sequentially. The animals were then euthanized, and the incision was extended. The (MMT/HA-GS)10 multilayer films modified and control PDMS sheets were washed and stained according to the kit protocol. After careful


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washing, the samples were sealed with tin foil and observed by fluorescence microscope investigation (Zeiss, Germany). After obtaining culture samples, each pocket was excised en bloc, and transmural sections from representative areas taken. Specimens were fixed in 10% formalin and embedded in paraffin blocks. Tissue sections, 5 mm thick, were mounted onto slides, which were stained using hematoxylin and eosin.

Statistical analysis All experiments were conducted in triplicate, and data points were expressed as the mean. Two sample t test in origin 8.0 (Microcal, USA) were used to compare data obtained with the different samples under identical treatments. A value of p < 0.05 was considered significant.

3. Results and discussion

3.1. Construction of the (MTT/HA-GS)10 Multilayer Film

LBL multilayer films can be constructed through alternate deposition of polymers with complementary noncovalent interactions such as electrostatic forces, hydrogen bond forces, hydrophobic interactions, van der Waals forces et al.. However, in this system, both MMT and HA carry negative charges in neutral environment. In order to circumvent electrostatic repulsion between MMT and HA, the pH of the self-assembly solutions was set at 2.5 during film deposition. At such pH, HA carry negative charges or in non-dissociation state (pKa of -COOH is ~2.9) and MMT carry



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positive charges as the protonation of -AlOH groups (pKa of AlOH2+ is ~5, pKa of AlOH and SiOH=7.9-8.5). In this way, the driving forces of MMT and HA-GS self-assembly included should be hydrogen bonding, electrostatic interactions and dipole–cation interactions. As indicated in Figure 2, the thickness of the (MMT/HA-GS)n multilayer films linearly grew to 429.8±42.5 nm for ten bilayers. Furthermore, SEM section images were also measured to examine the morphology and thickness of the multilayer films (Figure 4a). The thickness was uniform and the surface was quite rough with homogeneous gully structure. The rapid growth of the organic-inorganic hybrid multilayer films might be due to the increasing of roughness during film deposition.

Figure 2. Ellipsometry measurement of the (MMT/HA-GS)10 multilayer films. The MMT solution was 0.5mg/mL at pH=2.5 in water and HA-GS solution was 1 mg/mL and 0.5 mg/mL respectively at pH 2.5 in water. The experiment was conducted five times and data points were expressed as the mean.

3.2. Enzymatic

and

Bacterial

Responsive

Drug


Release

From

the

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(MMT/HA-GS)10 Multilayer Film Quantitative of GS release cannot be directly measured due to the absence of spectral absorption peak. The loading dosage and drug release behavior was determined by o-phthaldialdehyde dyeing at 332 nm. The calibration curve ranging from 0 to 200 µg/mL was obtained to calculate the GS concentrations. As shown in Figure S1, the Adj. R-Square was 0.99812 indicating the good linear relationship between GS concentration and UV absorbance. It was surprisingly that HAS also possess UV absorption at 332 nm, which also linearly grew with HAS concentration (R2=0.99827). The o-phthaldialdehyde also could react with the amino groups in HAS and the absorbance at 332 nm chromophoric products was obtained. Furthermore, the absorbance of HAS and GS mixture was the absorption sum of the two components. As a result, GS concentration was calculated through deducing the absorbance of the HAS. Furthermore, it is not an easy work to directly load small molecular weight hydrophilic antibiotics into multilayer thin films due to the rapid diffusion of the antibiotics. However, it has been proved that hydrogel-like MMT/PAA multilayer films could load high mount of GS which took up ~45% of the dry film matrix mass31. In the present work, the loading dosage of the (MTT/HA-GS)10 hybrid multilayer film was calculated through deducting the remaining GS in HA-GS solution from input GS amount. The measured loading dosage was 0.85±0.13 mg/cm2 which was much higher than many other reported multilayer films that were less than 200 µg /cm26, 16. Both of the ionic pairs formed between carboxylic groups of HA and protonated amino groups of GS and the good adsorption performance of MMT to positively



charged molecules should contribute to the high drug loading of GS.

The enzyme responsive GS release from the multilayer films was firstly tested in 88 U/mL and 175 U/mL HAS comparing with 0.1 M PBS as control (Figure 3a). It was found that GS released into PBS at the first 4 h and hardly release in the following 44 h indicating the strong retention property of MMT against GS release. At the presence of 88 U/mL HAS, the release of GS was promoted indicating the enzymatic release of the drug. Through adjusting the concentration of the HAS, the enzymatic release of GS responsive to HAS was greatly enhanced at the concentration of 175 U/mL (Figure 3a). It was reported that HA could be enzymatic degraded in a few hours at the presence of HAS showing the high sensitivity35, 48. However, in the GS loaded (MMT/HA-GS)10 systems, MMT could prevent diffusion of HAS as well as retention of GS release from the films. The actual structure of the multilayer films was not the simple stacking of layered assembled component A and B, but the interspersed structure45,

49, 50

. In this case, HAS in the solution could gradually

degrade the multilayer films in the top-to-down way. The degradation of the films might not only lead to the GS release and elimination of the pathogens, but also would resist pathogens adhesion through film peeling from the surface. As for the responsive GS release to bacteria, two kings of common pathogens with different concentration were used as model bacteria. As shown in Figure 3b, both E. coli and S. aureus infections microenvironment could promoted the GS release based on the HAS secretion and enzymatic degradation of the multilayer films. It is


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noteworthy that the drug loaded multilayer films were more responsive to E. coli than that to S. aureus and the responsiveness was concentration dependent. Especially as the concentration of E. coli was 105 CFU/mL, the release dose at 48 h increased 4 times comparing with that in PBS. As illustrated in Figure 4, the surface morphology and cross-sectional structure of the multilayer films during the enzymatic process were also explored by SEM measurements. Before drug release (Figure 4a and 4c), the film surface was very rough and the film thickness was uniform. As the degradation of the films for 48 h, there were more bump structures and porous structures throughout the films. The uniform of the degraded film indicated that the degradation mechanism of the film was peeling from the surface. Although after 48 h GS release in 105 CFU/mL E. coli, the total amount of released drug was nearly 30% of the total drug loaded in the multilayer films. The thickness of the multilayer film decreased from 429.8±42.5 nm to 389.3±34.7 nm and it also proved the presence of most of the GS in the films. We also tested the surface morphology and cross-sectional structure of the multilayer films after immersing in 0.1 M PBS for 48 h. However, there was no difference from the multilayer films before immersing which indicated the good stability of the films (data not shown). It can be inferred that more GS would release from the surface as the prolonging of the films enzymatic degradation.



Figure 3. GS release in (a) different concentration of HAS solution and (b) different concentration of S. aureus or E. coli solution.

Figure 4. SEM images of topographical features and the changes of thickness of the (MMT/HA-GS)10 multilayer films: (a), (c) before GS release, (b), (d) after GS release in 105 CFU/mL E. coli for 48 h

3.3. In vitro antibacterial test

Firstly, the bactericidal activity of the drug delivery system was examined through


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contacting with bacteria solutions and measured by flat counting method. The images were taken by camera and the number of bacterial colonies was counted in Figure 5c. It could be observed that all of the E. coli or S. aureus (~103 and 105 CFU/mL) were eliminated in 1 h indicating the rapid release of GS from the matrices. As shown in Figure 3, the released GS concentration was much higher than the minimal inhibition concentration of GS against E. coli and S. aureus which caused high efficacy bactericidal property. The LIVE/DEAD BacLight bacterial viability kit was also used to determine bactericidal and adhesion resistance properties of the multilayer films. As shown in Figure 6, a large number of bacteria stained in green color adhered on the control (glass) surface after incubation with 105 CFU/mL E. coli or S. aureus for 24 h. The lack of bactericidal component and antibiotics led to the absence of dead bacteria strained in red on the film surface. On the other hand, the total number of bacteria on the multilayer films surface including living and dead cells was much less than that on glass surface. For the adhered bacteria on the GS loaded multilayer films, most of the bacteria had been killed that stained with red color. The enzymatic degradation and rapid GS release contributed to the bacteria adhesion resistance and high efficient bactericidal properties. The peeling of HA and MMT components from the films can both resist bacteria adhesion and long-term biofilm inhibition. As the adhesion is the first but most decisive step in the process of bacterial infections development and biofilm formation. However, most of the as prepared antibacterial coatings on biomaterials cannot be eroded in a self-defense way. Most importantly, it is hard to remove the adhered



bacteria including living and dead in a self-cleaning way. In our previous study, multilayer films constructed based on hydrogen bonding could be tuned to be hydrolyzed in a controlled way to resist bacterial adhesion45. In the present work, the enzymatic degradation of HA component not only contributed to the GS release, but also led to the peeling of HA and MMT components from the films. Especially the peeling of lamellar structure of MMT might take away a whole piece of bacterial plaque or colonies. The self-defense bacterial infections responsive GS release together with removal of the bacterial body could long-term resist biofilm formation.

Figure 5. Bacterial colonies of the bacteria solutions (a, b) before or (a’, b’) after incubation with (MMT/HA-GS)10 multilayer film for 1h against E. coli or S. aureus and (c) number of bacteria colonies of the bacteria solutions

Figure 6. Fluorescent microscopy images of E. coli adhesions on (a, a’) glass, (c, c’) (MMT/HA-GS)10 multilayer film and S. aureus on (b, b’) glass and (d, d’)


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(MMT/HA-GS)10 multilayer film after incubation for 24 h

Zone of inhibition (ZOI) assays was used to detect the drug diffusion rate from the multilayer films based on enzymatic degradation. As shown in Figure 7, before immersing in PBS, the ZOI was 10.1±1.5 mm and 9.2±1.7 mm for S. aureus and E. coli respectively (the showed sample size was 0.53 cm2 and 0.72cm2, respectively). After being immersing in PBS for 24 h, the ZOI decreased to 8.6±1.2 mm and 8.1±0.8 mm for S. aureus and E. coli respectively (the showed sample size was 0.48 cm2 and 0.58cm2, respectively). Such large ZOI could be due to the enzymatic drug release from the multilayer films through incubation with the bacteria. For the control, there was no ZOI around the silicon wafer indicating the no antibacterial agent release. For the implants in human, the long-term antibacterial properties play an important role in resisting bacterial infections and biofilm formation. The ZOI of the drug delivery systems was measured after being immersing in PBS for 3, 5, 8 and 20 d against S. aureus and the result was showed in Figure 8. The ZOI gradually decreased from 8.4±2.5 mm to 8.2±2.0 mm, 7.6±1.8 mm and 6.2±1.3 mm respectively (the showed sample size was 0.48 cm2, 0.48 cm2, 0.78 cm2 and 0.34cm2, respectively). In this process, the multilayer films gradually degraded and more and more GS released in the solution. Fortunately, even after 20 d contacting with the bacterial solution, the tested ZOI was not much less than the just prepared multilayer films (data not shown). This proved that the high drug loading dosage was beneficial to the long-term antibacterial property.



Figure 7. Inhibition zones of (a, d) silicon wafer, (b, e) (MMT/HA-GS)10 multilayer films before immersing, (c, f) (MMT/HA-GS)10 multilayer films after being immersed in 0.1 M PBS 24 h against S. aureus and E. coli respectively, (g) Size of inhibition zone of the (MMT/HA-GS)10 multilayer films against the bacteria.

Figure 8. Inhibition zones of the (MMT/HA-GS)10 multilayer films after being immersed in 0.1 M PBS for (a) 3 d, (b) 5 d, (c) 8 d, (d) 20 d against S. aureus and (e) size of inhibition zone of the (MMT/HA-GS)10 multilayer films against S. aureus.

3.4. Antibacterial activity in vivo

Bacterial infection model in rabbits was used to explore the in vivo antibacterial property of the GS loaded multilayer films 6. Twenty four rabbits equally divided into


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two groups were implanted with either the antibacterial films coated or pristine PDMS. After being implanted with the antibacterial film modified or pristine PDMS, each pocket was inoculated with 0.2 mL, 108 CFU/mL S. aureus for simulated wound infections. After 7 d incubation, the implants were taken out for bacterial living/dead observation after fluorescent staining. The bacterial density on both pristine and antibacterial films modified PDMS were counted through ultrasonic cleaning and agar plate coating and counting. As shown in Figure 9a and 9a’, there were numerous living bacteria (87±16 CFU/104 µm2) on PDMS surface and nearly no dead bacteria (less than 3 CFU/104 µm2). There was no expected biofilm formation which could be owing to the shortage of nutrients transferring in subcutaneous non-flowing pocket. However, the pristine PDMS surface was almost covered with bacteria, it could be speculated that biofilm would inevitably form as the time going on or in other body fluid environments with nutrients. In comparison, there was sporadically few living bacteria (less than 2 CFU/104 µm2) but lots of dead bacteria on the multilayer films surface (27±7 CFU/104 µm2) which indicated the bactericidal property of the drug delivery systems. The number of living bacteria in the surrounding tissue was also examined through agar plate coating and counting of the leaching liquid. It showed that the number of living bacteria around multilayer films modified PDMS (89±24 CFU/mL) was much less than that around pristine PDMS(4.5±0.2×105 CFU/mL). The reason for the reduction of sterilization could be owing to the complexity of the in vivo environment. The adhesion of fibrin, fibronectin and platelets et al. blocked the



contacting of HAS in the microenvironment with the drug loaded multilayer films. The films degradation and drug release rates would be greatly delayed and the sterilization efficiency also would be reduced. We have tested the GS release rate in serum with 175 U/mL HAS and calculated the GS concentration after dialysis of macromolecules that might process amino groups. However, the GS release profile did not greatly decreased and the total released GS amount was about 82 wt% of that released in PBS at 48 h. So many kinds of proteins, platelets and enzymes, especially macrophages and other kinds of immune system reactions all contributed to the compromised antibacterial property in vivo. The appearance of the wound after being cut open was recorded (Figure 9b and 9e). It indicated that all of the twelve pockets implanted with pristine PDMS were filled with much pus showing the serious immune response and bacterial infections. As for the multilayer film modified PDMS, there was only one wound occurring slight infections with a small amount of pus. The sustained release of antibiotics around the implants reduced the proliferation of bacteria and the immune system attacking. Hematoxylin and eosin stained sections (H&E staining) of the tissues adhering to the implants was investigated to examine the tissue response to the implants after 7 d incubation. The tissue adhering to pristine PDMS was irregular and partly tattered (Figure 9c) indicating the acute inflammatory response. Also, there were lots of inflammatory cells in the tissues adhering to the pristine PDMS. On the other hand, the number of inflammatory cells in the tissues adhering to antibacterial coating modified implants were much less than in the tissues of pristine PDMS. The


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color of the tissues was vivid and appearance was more intact than that of pristine PDMS. In summary, the in vivo antibacterial tests proved the high efficacy of bactericidal properties and good biocompatibility of the GS loaded multilayer films.

Figure 9. Fluorescent microscopy images of (a, a’, d, d’) the S. aureus adhesions, (b, e) photographs of implant pockets and (c, f) images of hematoxylin and eosin stained sections of surrounding connective tissues of PDMS and (PAA/(PVP/CHI))10 multilayer film coated PDMS that were taken out at seven day.

4. Conclusions In the present work, (MMT/HA-GS)10 hybrid multilayer films were constructed through self-assembly method to directly load high dosage of small molecular antibiotics. The films grew fast in a linear manner which could be attributed to the increase of roughness. The obtained GS loaded multilayer films showed obvious enzyme responsive degradation and GS release at a certain concentration. Also the multilayer films showed higher enzymatic degradation rate responsive to E. coli than that to S. aureus. ZOI assays proved the high efficacy bactericidal function and long-term antibacterial properties of the multilayer films against the bacteria. The enzyme responsive on-demand self-defense drug release could enhance the drug



utilization and greatly reduced the development of bacterial resistance. The peeling of the films from surface caused by enzymatic degradation led to the resistance of bacteria adhesion and long-term biofilm formation. Antibacterial activity in vivo showed high bactericidal property and good biocompatibility of the GS loaded multilayer films. As a result, the obtained drug loaded multilayer films can be applied to surface modification of implants and biomedical devices.

Acknowledgements The National Natural Science Foundation of China (31771026, 51403158, 81271703), National Key R&D Program (2016YFC1101201), National Science and Technology Major Project (2014ZX09303301) and Science & Technology Program of Wenzhou (Y20160068, Y20160058, Y20140701) are greatly acknowledged.

Supporting Information: The calibration curve ranging from 0 to 200 µg/mL was obtained to calculate the GS concentrations. As shown in Figure S1, the Adj. R-Square was 0.99812 indicating the good linear relationship between GS concentration and UV absorbance. It was surprisingly that HAS also possess UV absorption at 332 nm, which also linearly grew with HAS concentration (R2=0.99827). The o-phthaldialdehyde also could react with the amino groups in HAS and the absorbance at 332 nm chromophoric products was obtained.

Reference 1.

Jia, Z. J., Xiu, P., Li, M., Xu, X. C., Shi, Y. Y., Cheng, Y., Wei, S. C., Zheng, Y. F., Xi, T. F., Cai, H., and Liu, Z. J. (2016) Bioinspired anchoring AgNPs onto micro-nanoporous TiO2 orthopedic coatings: Trap-killing of bacteria, surface-regulated osteoblast functions and host responses, Biomaterials 75, 203-222.


Page 26 of 35

Page 27 of 35 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

Biomacromolecules

2.

Chen, R. X., Willcox, M. D. P., Ho, K. K. K., Smyth, D., and Kumar, N. (2016) Antimicrobial peptide melimine coating for titanium and its in vivo antibacterial activity in rodent subcutaneous infection models, Biomaterials 85, 142-151.

3.

Hoque, J., Akkapeddi, P., Yadav, V., Manjunath, G. B., Uppu, D. S. S. M., Konai, M. M., Yarlagadda, V., Sanyal, K., and Haldar, J. (2015) Broad Spectrum Antibacterial and Antifungal Polymeric Paint Materials: Synthesis, Structure-Activity Relationship, and Membrane-Active Mode of Action, Acs Applied Materials & Interfaces 7, 1804-1815.

4.

Lim, K. Y., Chua, R. R. Y., Bow, H., Tambyah, P. A., Hadinoto, K., and Leong, S. S. J. (2015) Development of a catheter functionalized by a polydopamine peptide coating with antimicrobial and antibiofilm properties, Acta Biomaterialia 15, 127-138.

5.

Zhao, X., Wu, H., Guo, B., Dong, R., Qiu, Y., and Ma, P. X. (2017) Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing, Biomaterials 122, 34-47.

6.

Wang, B. L., Jin, T. W., Xu, Q. W., Liu, H. H., Ye, Z., and Chen, H. (2016) Direct Loading and Tunable Release of Antibiotics from Polyelectrolyte Multilayers To Reduce Bacterial Adhesion and Biofilm Formation, Bioconjugate Chem 27, 1305-1313.

7.

Wang, B. L., Liu, H. H., Wang, Z. F., Shi, S., Nan, K. H., Xu, Q. W., Ye, Z., and Chen, H. (2017) A self-defensive antibacterial coating acting through the bacteria-triggered release of a hydrophobic antibiotic from layer-by-layer films, Journal of Materials Chemistry B 5, 1498-1506.



8.

Page 28 of 35

Dai, X., Guo, Q., Zhao, Y., Zhang, P., Zhang, T., Zhang, X., and Li, C. (2016) Functional Silver Nanoparticle as a Benign Antimicrobial Agent That Eradicates Antibiotic-Resistant Bacteria and Promotes Wound Healing, Acs Applied Materials & Interfaces 8, 25798-25807.

9.

Bergek, J., Trojer, M. A., Uhr, H., and Nordstierna, L. (2016) Controlled release of a microencapsulated arduous semi-hydrophobic active from coatings: Superhydrophilic polyelectrolyte shells as globally rate-determining barriers, J Control Release 225, 31-39.

10.

Wang, B. L., Lin, Q. K., Jin, T. W., Shen, C. H., Tang, J. M., Hana, Y. M., and Chen, H. (2015) Surface modification of intraocular lenses with hyaluronic acid and lysozyme for the prevention of endophthalmitis and posterior capsule opacification, Rsc Advances 5, 3597-3604.

11.

Xiong, M. H., Li, Y. J., Bao, Y., Yang, X. Z., Hu, B., and Wang, J. (2012) Bacteria-Responsive Multifunctional Nanogel for Targeted Antibiotic Delivery, Advanced Materials 24, 6175-6180.

12.

Lu, L., and Unsworth, L. D. (2016) pH-Triggered Release of Hydrophobic Molecules from Self-Assembling Hybrid Nanoscaffolds, Biomacromolecules 17, 1425-1436.

13.

Zhuk, I., Jariwala, F., Attygalle, A. B., Wu, Y., Libera, M. R., and Sukhishvili, S. A. (2014) Self-Defensive Layer-by-Layer Films with Bacteria-Triggered Antibiotic Release, Acs Nano 8, 7733-7745.

14.

Hizal, F., Zhuk, I., Sukhishvili, S., Busscher, H. J., van der Mei, H. C., and Choi, C. H. (2015) Impact of

3D

Hierarchical

Nanostructures

on

the

Antibacterial

Efficacy

of

a

Bacteria-Triggered Self-Defensive Antibiotic Coating, Acs Applied Materials & Interfaces 7, 20304-20313.


Page 29 of 35 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

Biomacromolecules

15.

Sjollema, J., Dijkstra, R. J. B., Abeln, C., van der Mei, H. C., van Asseldonk, D., and Busscher, H. J. (2014) On-demand antimicrobial release from a temperature-sensitive polymer Comparison with ad libitum release from central venous catheters, J Control Release 188, 61-66.

16.

Xu, Q. W., Liu, H. H., Ye, Z., Nan, K. H., Lin, S., Chen, H., and Wang, B. L. (2017) Antimicrobial efficiency of PAA/(PVP/CHI) erodible polysaccharide multilayer through loading and controlled release of antibiotics, Carbohydrate Polymers 161, 53-62.

17.

Dong, P., Zhou, Y., He, W. W., and Hua, D. B. (2016) A strategy for enhanced antibacterial activity against Staphylococcus aureus by the assembly of alamethicin with a thermo-sensitive polymeric carrier, Chemical Communications 52, 896-899.

18.

Mauri, E., Rossi, F., and Sacchetti, A. (2016) Tunable drug delivery using chemoselective functionalization of hydrogels, Materials Science & Engineering C-Materials for Biological Applications 61, 851-857.

19.

Wang, B. L., Liu, H. H., Zhang, B. J., Han, Y. M., Shen, C. H., Lin, Q. K., and Chen, H. (2016) Development of antibacterial and high light transmittance bulk materials: Incorporation and sustained release of hydrophobic or hydrophilic antibiotics, Colloids and Surfaces B-Biointerfaces 141, 483-490.

20.

Kamachi, Y., Bastakoti, B. P., Alshehri, S. M., Miyamoto, N., Nakato, T., and Yamauchi, Y. (2016) Thermo-responsive hydrogels containing mesoporous silica toward controlled and sustainable releases, Materials Letters 168, 176-179.

21.

Li, Z. B., Yuan, D., Jin, G. R., Tan, B. H., and He, C. B. (2016) Facile Layer-by-Layer Self-Assembly toward Enantiomeric Poly(lactide) Stereocomplex Coated Magnetite



Nanocarrier for Highly Tunable Drug Deliveries, Acs Applied Materials & Interfaces 8, 1842-1853. 22.

Zhou, J., Horev, B., Hwang, G., Klein, M. I., Koo, H., and Benoit, D. S. W. (2016) Characterization and optimization of pH-responsive polymer nanoparticles for drug delivery to oral biofilms, Journal of Materials Chemistry B 4, 3075-3085.

23.

Wu, S. S., Huang, X., and Du, X. Z. (2015) pH- and redox-triggered synergistic controlled release of a ZnO-gated hollow mesoporous silica drug delivery system, Journal of Materials Chemistry B 3, 1426-1432.

24.

Min, J., Braatz, R. D., and Hammond, P. T. (2014) Tunable staged release of therapeutics from layer-by-layer coatings with clay interlayer barrier, Biomaterials 35, 2507-2517.

25.

Wang, B. L., Ye, Z., Tang, Y. H., Liu, H. H., Lin, Q. K., Chen, H., and Nan, K. H. (2016) Loading of Antibiotics into Polyelectrolyte Multilayers after Self-Assembly and Tunable Release by Catechol Reaction, Journal of Physical Chemistry C 120, 6145-6155.

26.

Perez-Anes, A., Gargouri, M., Laure, W., Van Den Berghe, H., Courcot, E., Sobocinski, J., Tabary, N., Chai, F., Blach, J. F., Addad, A., Woisel, P., Douroumis, D., Martel, B., Blanchemain, N., and Lyskawa, J. (2015) Bioinspired Titanium Drug Eluting Platforms Based on a Poly-beta-cyclodextrin-Chitosan Layer-by-Layer Self-Assembly Targeting Infections, Acs Applied Materials & Interfaces 7, 12882-12893.

27.

Pavlukhina, S., and Sukhishvili, S. (2013) Smart Layer-by-Layer Assemblies for Drug Delivery, In Smart Materials for Drug Delivery, Vol 2 (AlvarezLorenzo, C., and Concheiro, A., Eds.), pp 117-152.


Page 30 of 35

Page 31 of 35 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

Biomacromolecules

28.

Hu, N. S., Frueh, J., Zheng, C., Zhang, B., and He, Q. (2015) Photo-crosslinked natural polyelectrolyte multilayer capsules for drug delivery, Colloids and Surfaces a-Physicochemical and Engineering Aspects 482, 315-323.

29.

Wang, L., Ren, K. F., Wang, H. B., Wang, Y., and Ji, J. (2015) pH-sensitive controlled release of doxorubicin from polyelectrolyte multilayers, Colloids and Surfaces B-Biointerfaces 125, 127-133.

30.

Shukla, A., Fuller, R. C., and Hammond, P. T. (2011) Design of multi-drug release coatings targeting infection and inflammation, J Control Release 155, 159-166.

31.

Pavlukhina, S., Zhuk, I., Mentbayeva, A., Rautenberg, E., Chang, W., Yu, X., van de Belt-Gritter, B., Busscher, H. J., van der Mei, H. C., and Sukhishvili, S. A. (2014) Small-molecule-hosting nanocomposite films with multiple bacteria-triggered responses, Npg Asia Materials 6.

32.

Li, W., Sun, L. L., Pan, L. J., Lan, Z. P., Jiang, T., Yang, X. L., Luo, J. C., Li, R. H., Tan, L. Q., Zhang, S. R., and Yu, M. G. (2014) Dendrimer-like assemblies based on organoclays as multi-host system for sustained drug delivery, Eur J Pharm Biopharm 88, 706-717.

33.

Irwin, N. J., McCoy, C. P., Jones, D. S., and Gorman, S. P. (2013) Infection-Responsive Drug Delivery from Urinary Biomaterials Controlled by a Novel Kinetic and Thermodynamic Approach, Pharmaceutical Research 30, 857-865.

34.

Bailiang Wang, H. L., Zefeng Wang, Shuai Shi, Kaihui Nan, Qingwen Xu, Zi Ye and Hao Chen. (2017) A self-defensive antibacterial coating acting through the bacteria-triggered release of a hydrophobic antibiotic from layer-by-layer films, Journal of Materials Chemistry B 5, 9.



35.

Baier, G., Cavallaro, A., Vasilev, K., Mailander, V., Musyanovych, A., and Landfester, K. (2013) Enzyme Responsive Hyaluronic Acid Nanocapsules Containing Polyhexanide and Their Exposure to Bacteria To Prevent Infection, Biomacromolecules 14, 1103-1112.

36.

Li, Y., Liu, G., Wang, X., Hu, J., and Liu, S. (2016) Enzyme-responsive polymeric vesicles for bacterial-strain-selective delivery of antimicrobial agents, Angew. Chem. Int. Ed. 128, 1792-1796.

37.

Ninan, N., Forget, A., Shastri, V. P., Voelcker, N. H., and Blencowe, A. (2016) Antibacterial and Anti-Inflammatory pH-Responsive Tannic Acid-Carboxylated Agarose Composite Hydrogels for Wound Healing, Acs Applied Materials & Interfaces 8, 28511-28521.

38.

Traba, C., and Liang, J. F. (2015) Bacteria responsive antibacterial surfaces for indwelling device infections, J Control Release 198, 18-25.

39.

Lu, Y. M., Wu, Y., Liang, J., Libera, M. R., and Sukhishvili, S. A. (2015) Self-defensive antibacterial layer-by-layer hydrogel coatings with pH-triggered hydrophobicity, Biomaterials 45, 64-71.

40.

Wang, B. L., Han, Y. M., Lin, Q. K., Liu, H. H., Shen, C. H., Nan, K. H., and Chen, H. (2016) In vitro and in vivo evaluation of xanthan gum-succinic anhydride hydrogels for the ionic strength-sensitive release of antibacterial agents, Journal of Materials Chemistry B 4, 1853-1861.

41.

Wang, B. L., Jin, T. W., Han, Y. M., Shen, C. H., Li, Q., Lin, Q. K., and Chen, H. (2015) Bio-inspired terpolymers containing dopamine, cations and MPC: a versatile platform to construct a recycle antibacterial and antifouling surface, Journal of Materials Chemistry B 3, 5501-5510.


Page 32 of 35

Page 33 of 35 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

Biomacromolecules

42.

Craig, M., Altskar, A., Nordstierna, L., and Holmberg, K. (2016) Bacteria-triggered degradation of nanofilm shells for release of antimicrobial agents, Journal of Materials Chemistry B 4, 672-682.

43.

Deacon, J., Abdelghany, S. M., Quinn, D. J., Schmid, D., Megaw, J., Donnelly, R. F., Jones, D. S., Kissenpfennig, A., Elborn, J. S., Gilmore, B. F., Taggart, C. C., and Scott, C. J. (2015) Antimicrobial efficacy of tobramycin polymeric nanoparticles for Pseudomonas aeruginosa infections in cystic fibrosis: Formulation, characterisation and functionalisation with dornase alfa (DNase), J Control Release 198, 55-61.

44.

Tian, Z., Zhang, Y., Liu, X., Chen, C., Guiltinan, M. J., and Allcock, H. R. (2013) Biodegradable polyphosphazenes containing antibiotics: synthesis, characterization, and hydrolytic release behavior, Polymer Chemistry 4, 1826-1835.

45.

Wang, B.-l., Ren, K.-f., Wang, H. C. J.-l., and Ji, J. (2013) Construction of Degradable Multilayer Films for Enhanced Antibacterial Properties, Acs Applied Materials & Interfaces 5, 4136-4143.

46.

Cado, G., Aslam, R., Seon, L., Garnier, T., Fabre, R., Parat, A., Chassepot, A., Voegel, J. C., Senger, B., Schneider, F., Frere, Y., Jierry, L., Schaaf, P., Kerdjoudj, H., Metz-Boutigue, M. H., and Boulmedais, F. (2013) Self-Defensive Biomaterial Coating Against Bacteria and Yeasts: Polysaccharide Multilayer Film with Embedded Antimicrobial Peptide, Advanced Functional Materials 23, 4801-4809.

47.

Sampath, S. S., and Robinson, D. H. (1990) Comparison of New and Existing Spectrophotometric Methods for the Analysis of Tobramycin and Other Aminoglycosides, Journal of Pharmaceutical Sciences 79, 428-431.



48.

Bean, J. E., Alves, D. R., Laabei, M., Esteban, P. P., Thet, N. T., Enright, M. C., and Jenkins, A. T. A. (2014) Triggered Release of Bacteriophage K from Agarose/Hyaluronan Hydrogel Matrixes by Staphylococcus aureus Virulence Factors, Chem Mater 26, 7201-7208.

49.

Hu, X. F., and Ji, J. (2011) Covalent Layer-by-Layer Assembly of Hyperbranched Polyether and Polyethyleneimine: Multilayer Films Providing Possibilities for Surface Functionalization and Local Drug Delivery, Biomacromolecules 12, 4264-4271.

50.

Shen, L. Y., Chaudouet, P., Ji, J. A., and Picart, C. (2011) pH-Amplified Multilayer Films Based on Hyaluronan: Influence of HA Molecular Weight and Concentration on Film Growth and Stability, Biomacromolecules 12, 1322-1331.


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