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May 31, 2015 - Anna Galperin, Karen Smith, Neil S. Geisler, James D. Bryers, and Buddy D. Ratner*. Department of Bioengineering, University of Washing...
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Precision-Porous PolyHEMA-Based Scaffold as an AntibioticReleasing Insert for a Scleral Bandage Anna Galperin, Karen Smith, Neil S. Geisler, James D. Bryers, and Buddy D. Ratner* Department of Bioengineering, University of Washington, 3720 15th Avenue NE, Seattle, Washington 98195, United States ABSTRACT: Combat-related penetrating ocular injuries have become a common form of battlefield injury in modern warfare and can lead to potentially devastating visual impairments. Prompt stabilization of the wounded globe via prevention of infection and fibrosis enhances the probability of a successful outcome after professional medical treatment. In this study, a norfloxacin-releasing poly(hydroxyethyl methacrylate)based insert was designed and fabricated as a part of scleral bandage to prevent development of infection and scar formation. First, a spheretemplating technique was applied, during which 2-hydroxyethyl methacrylate monomer was photocopolymerized with acrylic acid and/ or 4-fluorostyrene at different molar ratios to generate poly(hydroxyethyl methacrylate)-based porous scaffolds of various compositions with interconnected, monodisperse, 38 μm diameter pores. The scaffolds were then loaded with norfloxacin via swelling in drug-saturated solutions of various solvents, such as water, acetone, chloroform and ethanol, and the effect of the scaffold composition and the swelling solvent on norfloxacin uptake was explored. An in vitro drug release study was then conducted to explore the release kinetics of norfloxacin from the drug-loaded scaffolds, with the aim to find the optimal scaffold composition to provide release of norfloxacin over a 1 week period. The antibacterial potential of the optimal composition norfloxacin-loaded scaffold to inhibit the growth of the relevant clinical pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa during a 1 week period was evaluated in vitro using a continuous culture flow cell system and a soft agar overlay plate assay. KEYWORDS: porous hydrogel, 2-hydroxyethyl methacrylate, 4-fluorostyrene, scleral bandage, antibiotic release, infection, healing, antibacterial, Staphylococcus epidermidis, Pseudomonas aeruginosa

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INTRODUCTION Eye injuries have become one of the most common forms of battlefield injury in recent warfare as a result of modern weaponry that unleashes an explosive cascade of fragments (e.g., improvised explosive devices (IED) blasts) causing globe penetrating wounds with intraocular insertions of foreign bodies (FB).1−3 Where penetrating combat-related ocular injury occurs in the field, clean conditions and professional medical assistance for removal of intraocular FB and reconstructive surgery will probably not be available immediately. Infection often rapidly occurs and fibrotic healing is observed. These complications associated with penetrating scleral injuries often lead to severe visual impairment and even loss of vision.1,4−8 The appropriate management of the wounded globe as soon as possible to the time of injury is vital for successful outcome of forthcoming professional medical assistance.1 We have been working on development of a scleral bandage that could be applied immediately to the injured eye on the battlefield. The bandage is designed to seal the wounded globe, promote nonfibrotic healing, and prevent development of infection before advanced medical treatment can be implemented. The scleral bandage consists of a muco-adhesive contact lens with a pro-healing, antibiotic releasing insert. The insert is intended to prevent the development of fibrotic healing © XXXX American Chemical Society

thus reducing scar formation and to prevent the development of infection through topical delivery of an antibiotic drug. The work reported here is on the development of the pro-healing, antibiotic-releasing insert with particular emphasis on antibiotic release. We have previously reported the development of porous templated polymers (PTPs) based on synthetic biomaterials, such as poly(hydroxyethyl methacrylate) (polyHEMA), poly(N-isopropylacrylamide) (polyNIPAM), silicone elastomer, fibrin, and polyurethane that are characterized by an interconnected, monodisperse pore microstructure.9−14 In previous studies, PTPs with a pore diameter about 38 μm had demonstrated a superior healing in numerous soft and hard tissue applications through maximized vascularization and minimized fibrosis, probably due to directing the polarization of macrophages (MØ) toward a pro-healing M2 phenotype.15−22 Norfloxacin (NF) is a fluorquinolone antibiotic with broadspectrum activity against Gram-positive and Gram-negative bacteria, including Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa, and it is a component of Received: March 16, 2015 Accepted: May 31, 2015

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Discs were shaken at 25 °C for 48 h to allow the hydrogels to reach equilibrium swelling. Then discs were rinsed with the pure solvent that was used for the drug loading. Materials loaded with NF in water and organic solvents were then lyophilized or dried under vacuum, respectively. Each composition was prepared in triplicate. The normalized amount of encapsulated NF (mg/gDRY PTP) was calculated using eq 1:

commercially available eye drops CHIBROXIN used to treat and prevent eye infections.23,24 Here in we report fabrication of NF-releasing polyHEMAbased PTPs with a pore diameter of 38 μm. HEMA monomer was copolymerized with acrylic acid (AA) and/or 4fluorostyrene (FS) to find the optimal composition of the polyHEMA-based PTP that allowed for efficient NF incorporation and prolonged release during 7 days, which is a reasonable period of time for prophylaxis after many surgical procedures or for a wounded soldier to gain access to professional medical treatment. The antibacterial potential of the drug-loaded PTP was evaluated during a 1 week period against the troublesome clinical pathogens S. epidermidis and P. aeruginosa using a continuous culture flow cell system and an agar overlay plate assay.

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normalized encapsulated NF (mg/g PTP) = [(W2 − W1)/W1]1000

Where W1 and W2 are weights (mg) of 10 × 1 mm dry polyHEMAbased PTP discs before and after drug loading, respectively. NF Release Study. Dry, 10 × 1 mm polyHEMA-based PTPs discs of various compositions loaded with NF were placed in 3 mL of PBS buffer and shaken at 37 °C. Each composition of PTP disc was tested in triplicate. At each time point, a 3 mL aliquot of the PBS solution was withdrawn and replaced with a fresh 3 mL of PBS. The concentration of NF was measured using a UV−vis plate reader (SAFIRE II, Tecan Systems Inc., San Jose, CA) at 273 nm. The concentration of the released NF was calculated from a standard curve of the drug UV absorbance versus drug concentration in PBS and the mass of the drug in the known volume of the solution was quantified. In Vitro Antibacterial Studies. Bacterial Strains and Culture Conditions. Staphylococcus epidermidis strain RP62A (ATCC 35984) and Pseudomonas aeruginosa strain PA01 (ATCC 47085) were selected to test the efficacy of NF-loaded PTPs against a Gram-positive bacterial isolate and a Gram-negative bacterial isolate, respectively. Both isolates were grown on Tryptic Soy Agar (TSA) (Remel, KS, USA) and in liquid culture in Tryptic Soy Broth (TSB) (Remel). Minimum Inhibitory Concentration (MIC) of Norfloxacin. The minimum inhibitory concentration (MIC) of norfloxacin was determined for S. epidermidis strain RP62A and P. aeruginosa strain PA01 using a standard broth microdilution method developed by the Clinical and Laboratory Standards Institute (CLSI). A dilution series of the antibiotic was prepared in cation-adjusted Mueller-Hinton broth and incubated for 24 h in the wells of a 96-well microtiter plate with a known concentration of bacteria, adhering to the accepted standard methodology (CLSI document M07-A9, 2012).25 Following incubation, the minimum concentration of norfloxacin that inhibited growth of the bacteria was determined by examining the optical density of the wells at 600 nm. Antibacterial Activity of Drug-Loaded PTPs in a Continuous Flow System. A coupon evaluation flow cell (BioSurface, Montana, USA) system was used to test the efficacy of drug-loaded scaffold discs in comparison to drug-free control scaffold discs (in triplicate) under the clinically relevant continuous flow of nutrients.26 Scaffolds within the flow cell chamber were inoculated with 2 × 103 CFU/mL of either S. epidermidis (RP62A) or P. aeruginosa (PA01) and bacterial cells were allowed to adhere to the surface of the scaffold discs for 2 h. Fresh sterile TSB medium was then continuously pumped through the system (1 mL/min) for 1, 3, and 7 days. At these time points the scaffolds were removed from the flow cells, rinsed twice with PBS, sonicated, and vortexed (3 × 30s cycles) to remove bacterial cells from the surface, and then plated on TSA to perform colony counts. The survival (%) of the bacteria on drug-loaded scaffolds was calculated relative to the control drug-free scaffolds. NF-free and NF-loaded polyHEMA-based PTPs were visualized using scanning electron microscopy (SEM) (FEI SEM XL Siron, Hillsboro, OR) after 7 days incubation with bacteria in the flow cell system. PTPs were removed from the flow cell chambers, fixed in methanol:acetic acid (9:1) solution at 4 °C for 18 h, washed with water, snap-frozen in liquid nitrogen and lyophilized. The dry samples were Au/Pd sputter-coated for 60 s (SPI Supplies, West Chester, PA). Antibacterial Activity of Drug-Loaded PTPs in a Static Overlay Plate Assay. To test the kinetics of drug-release from the scaffold, a drug-loaded scaffold disc was placed in the center of a TSA plate (drug-free scaffolds were used as negative controls). Overnight cultures of S. epidermidis and P. aeruginosa were diluted in fresh TSB

MATERIALS AND METHODS

Materials. HEMA (ophthalmologic grade, 99%), acrylic acid (99%), 4-fluorostyrene (99%) and norfloxacin were purchased from Sigma (St. Louis, MO, USA), tetraethylene glycol dimethacrylate (TEGDMA) (Polyscience Inc.), uncross-linked poly(methyl methacrylate) (PMMA) microspheres of sphere diameter of 38 μm (Microbeads Inc.), HPLC grade dichloromethane, acetone, chloroform and ethanol were purchased from EMD Chemicals Inc. (Billerica, MA, USA), 2,2-dimethoxy-2-phenylacetophenone (IRGACURE 651, Ciba-Geigy). Methods. Fabrication of PolyHEMA-Based PTPs. Step 1. Template Preparation. PMMA microspheres (38 μm diameter) were transferred to a mold composed of two microscope slides separated by a 1 mm thick Teflon spacer and sonicated for 10 min to tightly pack the uniformly sized spheres. The beads were then sintered for 24 h at 175 °C to obtain PMMA templates with neck (interconnect) sizes of 30% of the bead diameter. Step 2. PTPs Fabrication. To fabricate PTPs of various compositions (Table 1), PMMA templates from step 1 were

Table 1. Compositions of PolyHEMA-Based Hydrogels monomers abbreviation of hydrogel composition

HEMA

poly(HEMA-AA) poly(HEMA-AA-0.5FS) poly(HEMA-AA-1.5FS) poly(HEMA-AA-2.5FS) poly(HEMA-2.5FS)

+ + + + +

AA, % molAA/ molHEMA 2.5 2.5 2.5 2.5

(1)

FS,% molFS/ molHEMA 0.5 1.5 2.5 2.5

infiltrated with a reaction mixture composed of HEMA (1 g, 7.8 × 10−2 mol), appropriate amount of AA and/or FS (molar ratios are given in Table 1), TEGDMA (48 μL, 1.5 × 10−4 mol, 2% mol/molHEMA) and UV initiator IRGACURE 651 (5 mg, 1.9 × 10−5 mol, 0.25% mol/molHEMA) in water (1 mL). PMMA templates infused with the monomer mixture were photopolymerized for 5 min from each side using a UV lamp (PC451050, Hanovia 450-W Hg lamp, HANOVIA Specialty Lighting LLC, NJ). Distance of the lamp from the sample was 30 cm. Then PMMA-hydrogel composite was removed from the mold and placed in dichloromethane to dissolve the PMMA beads. The polyHEMA-based PTPs of various compositions (Table 1) were washed in acetone and then hydrated in distilled water. Encapsulation of NF in PolyHEMA-Based PTPs. Dry discs (10 mm × 1 mm) of PolyHEMA-based PTPs of various compositions (Table 1) were placed in 10 mL of following NF solutions: water (0.28

mgNF/mL), ethanol, acetone and chloroform (2 mgNF/mL). B

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ACS Biomaterials Science & Engineering (1/100) and incubated at 37 °C, with shaking at 180 rpm, until an OD600 = 0.6 was achieved. A 100 μL sample of each culture was then added to 5 mL of molten soft agar (0.7% w/v agar in sterile water) and poured over the TSA plate on top of the scaffold disc. The top layer was allowed to solidify for 5 min and then the plates were incubated for 18h at 37 °C. Each day the scaffold disc was removed from the plate and used to set up a new overlay plate, exactly as described previously. The zones of inhibition produced were evaluated each day until no inhibition was observed.

toward the polyHEMA network; (b) polyFS is hydrophobic and its presence can reduce the overall hydrophilicity of polyHEMA-based network and thus slow the drug release rate.33 Table 1 summarizes polyHEMA-based hydrogels that were synthesized at various monomer molar ratios to explore the effect of polyAA and polyFS fractions on encapsulation efficacy and release profile of NF. To fabricate polyHEMA-based PTPs of various compositions (Table 1), we applied the sphere templating technique. The technique forms a cross-linked polymer matrix around a sacrificial template of sinter-fused, monodisperse PMMA microspheres and then removing the template to generate a porous biomaterial.9 This specific approach to spheretemplating allows for the fabrication of a monodisperse, highly interconnected PTP with defined pore size.9−11 In this study, monodisperse PMMA microspheres of 38 μm diameter are introduced into the mold, sintered to fuse particles, infiltrated with monomer mixture of HEMA, AA and/ or FS, TEGDMA, and photoinitiator, polymerized in situ to obtain a composite of polyHEMA-based hydrogel and PMMA particles, and finally exposed to solvent to solubilize the PMMA particles (Figure 2). This yields highly interconnected PTPs with pore diameter of 38 μm that is optimal to promote nonfibrotic healing of damaged sclera. The typical morphology of a polyHEMA-based PTP is shown in Figure 3. Drug Loading Study. NF is a UV sensitive molecule and therefore could not be encapsulated within polyHEMA-based PTPs in situ, during the fabrication since UV is used to initiate the polymerization. Therefore, the aim of this study was to load the PTPs with NF through immersion and swelling of dry discs of PTPs in near saturated solution of the drug in various solvents (water, acetone, ethanol and chloroform) thus incorporating NF. The goal of the drug loading study was to explore the effect of the composition of the PTP and the type of swelling solvent on the encapsulation of NF. Figure 4 presents the normalized loading from various swelling solvents of encapsulated NF (mg) per gram of dry PTP. In all swelling solvents, the amount of NF encapsulated in PTPs that contain AA is higher in comparison to amount of encapsulated NF in poly(HEMA-2.5FS), the PTP without AA in its composition. The results are reasonable since it was reported that NF possess great affinity toward AA.32 Also, PTTs with AA are more hydrophilic compared to poly(HEMA-2.5FS) and therefore are more swollen leading to more efficient encapsulation. Additionally, Figure 4 demonstrates the same tendency for PTPs of all compositions: the amount of NF encapsulated using a saturated solution of NF in organic solvents is higher than the amount of NF encapsulated using an aqueous saturated solution of the drug. This difference could be explained by the significant difference in NF solubility, which is ten times more soluble (2 mgNF/mL) in the organic solvents used in this study (chloroform, acetone, and ethanol) compared to water (0.28 mgNF/mL). As part of scleral bandage, the polyHEMA-based insert will have an average weight of 17 mg. Taking into account that the concentration of NF in CHIBROXIN is 3 mg/mL with an average dose of 2 drops × 4 times/day, where the volume of one drop is approximately 25 μL and bioavailability of the drug is approximately 5%, the daily amount of the drug that penetrates the cornea and provides therapeutic effect is 0.03 mg.34,35 Therefore, to show an antibacterial effect over 1 week

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RESULTS AND DISCUSSIONS Design and Fabrication of PolyHEMA-Based PTPs. There are two main compositional requirements for the PTP for it to serve as an antibiotic releasing insert in a scleral bandage: (a) the material should possess good affinity for NF in order to encapsulate a therapeutically active concentration of the drug and retain antibacterial activity for at least 1 week; (b) the material should allow for sustained drug release over a 1 week period to deliver the antibiotic at a concentration that will prevent infection. PolyHEMA was chosen as a material for the insert since polyHEMA-based hydrogels are widely used for biomedical applications and have an extensive history of use, particularly for ophthalmic applications.27−29 PolyHEMA hydrogels meet many of the criteria set for this insert because it is a soft, flexible, water-absorbing, relatively inert to biological processes, resistant to degradation, can be fabricated to appropriate shape, and is easily sterilized. However, polyHEMA alone could not be loaded with sufficient NF to provide prolonged release of the drug.30−32 In previous studies, we reported on polyHEMA hydrogels that were surface-modified with octadecyl isocyanate to produce a hydrophobic rate-limiting barrier controlling NF release to prevent infections after cataract surgery.30,31 In this work we modified the bulk composition of polyHEMA in order to design a polyHEMA hydrogel with desired drug release properties. HEMA monomer was copolymerized with AA and FS monomers (Figure 1) to create polyHEMA-based hydrogels

Figure 1. Chemical structure of NF and of the monomers included in the design of the scleral insert.

of various compositions (Table 1). HEMA was copolymerized with AA since Alvarez-Lorenzo et al. had reported that incorporation of polyAA dramatically increases affinity of NF to polyHEMA hydrogel.32 The rationale for the incorporation of FS in polyHEMA has two parts: (a) there is a similarity between the structure of the FS and the part of the NF molecule with a fluorine atom attached to the aromatic ring (Figure 1). This may positively impact the affinity of the drug C

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Figure 2. Fabrication of polyHEMA-based PTPs.

Figure 3. SEM images of polyHEMA-based scleral insert at various magnifications.

2.5FS) was obtained with ethanol as a drug-loading solvent, which released NF over a two week period, exceeding the 1 week target (Figure 5-C). Interestingly, the presence of the increasing FS fraction in AA containing polyHEMA-based PTPs does not have a pronounced effect on the drug release profile in comparison to the poly(HEMA-AA) PTP. The swelling study in PBS indicated that an increase in FS does not affect significantly swelling properties of the poly(HEMA-AA) PTPs, while the swelling % of poly(HEMA-2.5 FS) PTP is 1.7 fold lower in comparison to poly(HEMA-AA): 92 ± 10 and 156 ± 11%, respectively. The results of the swelling study correlate with the drug release profile showing that addition of FS does not significantly decrease the water content of the poly(HEMA-AA) network, whereas in the absence of AA, FS had a pronounced effect, making the polyHEMA network more hydrophobic and therefore reducing the diffusivity of the NF. To further explore the effect of FS on the release of NF from polyHEMA-based PTPs, we doubled the fraction of FS, fabricated poly(HEMA-5FS) PTP, and loaded it with the drug in ethanol. Figure 5-C demonstrates an additional decrease in NF release profile in comparison to poly(HEMA2.5FS), probably due to an increase in hydrophobic nature of the material. In Vitro Studies. Antibacterial Activity of Drug-Loaded Scaffolds in a Continuous Flow System. First, 10 × 1 mm discs of drug-loaded and drug-free poly(HEMA-2.5FS) PTPs were inoculated with either S. epidermidis RP62A (Grampositive) or P. aeruginosa PA01 (Gram-negative). The bacteria were allowed to adhere to the surface of the discs and then exposed to a continuous flow of fresh media for 1, 3, and 7 days. At each time point, the bacterial growth on the drugloaded and drug-free PTP was ascertained by performing viable colony counts. The inhibition of bacterial growth (%) by drugloaded samples was calculated by comparing the bacterial

Figure 4. Normalized amount of encapsulated Norfloxacin in polyHEMA-based scaffolds loaded with the drug in various swelling solvents.

the insert should encapsulate at least 0.21 mg of NF. Calculation of the amount of the drug per insert based on normalized data displayed in Figure 4 shows that polyHEMAbased PTPs of all compositions and in all loading solvents encapsulate enough NF to provide a therapeutic effect over a 1 week period. Drug Release Study. Figure 5 demonstrates release of NF from polyHEMA-based PTP’s of various compositions that were loaded with NF in different swelling solvents. For all drugloading solvents the same pattern emerged: the PTPs with AA in their structure released the drug after approximately 2 days, whereas poly(HEMA-2.5FS), the PTP without AA that contains FS in the composition, displayed prolonged drug release. The optimal drug release profile for poly(HEMAD

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Figure 5. Release profiles of NF from polyHEMA-based scaffolds of various compositions loaded with the drug in (A) water, (B) chloroform, and (C) ethanol. Arrow is pointing at one-week time point.

Table 2. Inhibition of Bacterial Growth (%) on Drug-Loaded PTP Discs in Comparison to Drug-Free Controls during Continuous Culturea inhibition of growth (%) S. epidermidis poly(HEMA-2.5FS) poly(HEMA-AA-2.5FS) a

P. aeruginosa

day 1

day 3

day 7

day 1

day 3

day 7

98.7 ± 0.5 N/A

95.8 ± 2.4 N/A

81.5 ± 5.2 N/A

98.6 ± 0.3 N/A

95.0 ± 0.5 N/A

72.8 ± 3.6 6.2 ± 5.5

To quantitate the bacteria population, bacteria were removed by sonication and vortexing, as detailed in the Experimental Section.

counts to those recovered from the drug-free control samples and the results are summarized in Table 2. As presented in Table 2, NF loaded poly(HEMA-2.5FS) PTP inhibits 98.7, 95.8 and 81.5% of S. epidermidis growth during 1, 3, and 7 days in the flow cell system, respectively, and 98.6, 95.0 and 72.8% of P. aeruginosa growth during 1, 3, and 7 days in the flow cell system, respectively. These results indicate that despite continuous flow of the fresh medium through the PTP, the drug was released in a sustained manner allowing for the effective inhibition of the bacterial growth during the desired one-week period. This correlates with our drug release kinetic study that showed the sustained release of NF from poly(HEMA-2.5FS) scaffolds over at least 7 days (Figure 1). There are differences in inhibition (%) of cell viability after 7 days between S. epidermidis and P. aeruginosa (81.5% and 72.8%, respectively). This is as expected due to the marked structural differences between these Gram-positive and Gramnegative bacterial strains. When the minimum inhibitory

concentration (MIC) of norfloxacin was tested for these two strains, it was found that the S. epidermidis strain had a lower MIC than the P. aeruginosa strain, of 0.3 and 4.3 μg/mL, respectively, which correlates with the slight reduction in inhibiton (%) of P. aeruginosa cells in comparison to S. epidermidis after 7 days. The 7 day flow cell experiment was also performed with NFloaded poly(HEMA-AA-2.5FS) PTP that contains in its composition, besides FS (the same molar ratio as in poly(HEMA-2.5FS)), also acrylic acid (AA). According to the drug release profile presented in Figure 5, poly(HEMA-AA2.5FS) releases close to 100% of the drug by day 2 and therefore we expected to see low, if any, inhibition of the bacterial growth after 7 days in the flow cell system. Our assumption was supported by a small 6.2% ± 5.5 inhibition of P. aeruginosa growth for poly(HEMA-AA-2.5FS) after 7 days in the flow cell system. Again, this result correlates with the drug release profile reported in Figure 5 and serves as an additional E

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Figure 6. Representative SEM images of poly(HEMA-2.5FS) PTP inoculated with an S. epidermidis strain after 7 days in the flow cell. (A, B) Drugfree PTPs (controls); (C, D) drug-loaded scaffolds.

Figure 7. Representative photos of TSA plates seeded with either S. epidermidis or P. aeruginosa strains and incubated with drug-loaded poly(HEMAAA), poly(HEMA-AA-2.5FS) ,and poly(HEMA-2.5FS) PTPs during the soft agar overlay plate experiment. The zones of inhibition produced, presented as dark circles, were evaluated each day until no inhibition was observed.

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approximately 2 days, while the poly(HEMA-2.5FS) scaffold released the drug during the desired 7 days period (Figure 5). This previously reported difference in drug release kinetic is supported by the results of the soft agar overlay plate experiment and supports the conclusion that poly(HEMA2.5FS) is an optimal scaffold composition for desirable 7 days slow release of the drug and effective inhibition of the bacterial growth.

control, besides the drug-free poly(HEMA-2.5FS) scaffold. Despite the fact that the normalized amount of encapsulated NF in poly(HEMA-2.5FS) is approximately 2-fold lower than in poly(HEMA-AA-2.5FS) [40 ± 7 and 93 ± 19 mg(drug)/ g(dry PTP), respectively (Figure 4)] the poly(HEMA-2.5FS) shows efficient bacterial growth inhibition during the 7 days period because of the slow release of the drug over time. The flow cell experiment is a dynamic study, since the drug is continuously washed away from the PTP during 7 days, loosely analogous to the tear flow in the eye. In the setting in which this scleral insert will be used we anticipate that our PTP-based scleral insert will be more efficient since the natural tear fluid circulation in the eye is significantly slower compared to the more extreme conditions of the flow cell system, especially if the eye will be sealed with the scleral bandage. Figure 6 offers visual confirmation of the results discussed above showing representative SEM images of the drug-free and drug-loaded poly(HEMA-2.5FS) scaffold after 7 days of the flow cell experiment conducted with the S. epidermidis strain. It can be seen that surface of the drug-free scaffold is almost completely covered with S. epidermidis (Figure 6A) after 7 days in the flow cell in contrast to the relatively bacteria-free surface of the drug- loaded scaffold (Figure 6C). Greater magnification shows densely packed S. epidermidis cocci cells growing in a multilayered biofilm structure on the drug-free scaffold while the surface of the drug-loaded scaffold (Figure 6D) shows mostly sparsely scattered single bacterial cells. The same tendency was observed for the drug-free and drug-loaded scaffolds inoculated with P. aeruginosa after 7 days in the flow cell. The SEM images offer visual support of the flow cell experiment quantitative results presented in Table 2. Antibacterial Activity of Drug-Loaded Scaffolds in a Static Soft Agar Overlay Assay. The application for our antibioticreleasing insert is as part of a wound dressing that will remain in place on the injured eye until professional medical assistance can be obtained. In the previous continuous culture experiment the drug-loaded scaffolds were subjected to constant washing with media but we also wanted to examine the effect of drug release on bacteria in a static system. The drug-loaded scaffold (poly(HEMA-AA), poly(HEMA-AA-2.5FS) and poly(HEMA2.5FS)) was placed on an agar plate and covered with an even layer of bacteria in soft agar which then solidified. After 24 h of incubation, plates were examined for a zone of clearing in the bacterial layer and this was measured and photographed. The disc was removed each day and placed on a new plate with a fresh bacterial culture until no inhibition was observed (drugfree scaffolds were used as negative controls). Figure 7 presents representative images of TSA plates incubated with drug-loaded scaffolds. For the drug-loaded poly(HEMA-AA) and poly(HEMA-AA-2.5FS) PTPs inhibition of S. epidermidis was detected over 5 days. The zone of inhibition decreased with time, while no inhibition was observed at day 7. For P. aeruginosa no inhibition was detected at day 5 for the drug-loaded poly(HEMA-AA) and poly(HEMA-AA-2.5FS) PTPs. However, for the drug-loaded poly(HEMA-2.5FS) PTP, inhibition of S. epidermidis and P. aeruginosa was observed for times as long as 11 and 7 days, respectively. As was mentioned above, the PTP that contains AA took up significantly more NF than PTP with only FS because of the increased affinity of the drug to AA (Figure 3). However, due to increased hydrophilicity associated with the presence of AA, the drug release from poly(HEMA-AA) and poly(HEMA-AA-2.5FS) scaffolds was fast and ended after

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CONCLUSIONS The results reported here show that despite the fact that polyAA-containing PTPs are more efficient in NF incorporation, the presence of AA results in a more rapid drug release profile, probably due to increased hydrophilicity of the polyHEMA-based hydrogel. FS containing PTPs, in absence of AA, show prolonged release over the desired one-week period. We assume that FS slows the release of NF not only by decreasing the overall hydrophilicity of polyHEMA-based network but also by retaining the drug within the hydrogel network due to a similarity in structure, i.e., a fluorine atom attached to an aromatic ring. In vitro continuous flow cell and static studies correlate with the drug release profile and demonstrate that the optimal poly(HEMA-2.5FS) PTP encapsulates and releases the drug at levels that provide an antibacterial effect over a period of 7 days. Future studies will focus on the in vivo evaluation of the antibacterial and prohealing potential of the poly(HEMA-2.5FS) insert.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Telemedicine & Advanced Technology Research Center (TATRC) at U.S. Army Medical Research and Materiel Command (USAMRMC). We thank Daniel Fisher for technical help during the antibacterial study.

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REFERENCES

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DOI: 10.1021/acsbiomaterials.5b00133 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.5b00133 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX