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. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: Int Forum Allergy Rhinol. 2019 Nov 6;10(1):121–127. doi: 10.1002/alr.22475

In vitro Evaluation of a Ciprofloxacin and Azithromycin Sinus Stent for Pseudomonas aeruginosa Biofilms

Dong-Jin Lim 1, Daniel Skinner 1, John Mclemore 1, Nick Rivers 1, Jeffrey Brent Elder 1, Mark Allen 1, Connor Koch 1, John West 1, Shaoyan Zhang 1, Harrison M Thompson 1, Justin P McCormick 1, Jessica W Grayson 1, Do-Yeon Cho 1,2,*, Bradford A Woodworth 1,2,*
PMCID: PMC6942221  NIHMSID: NIHMS1055741  PMID: 31692289

Abstract

Background:

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease characterized by persistent inflammation and bacterial infection. Ciprofloxacin and azithromycin are commonly prescribed antibiotics for CRS, but the ability to provide targeted release in the sinuses could mitigate side effects and improve drug concentrations at the infected site. This study was aimed to evaluate the efficacy of the novel ciprofloxacin-azithromycin sinus stent (CASS) in vitro.

Methods:

The CASS was created by coating ciprofloxacin (hydrophilic, inner layer) and azithromycin (hydrophobic, outer layer) onto a biodegradable poly-L-lactic acid (PLLA) stent. In vitro evaluation included: 1) assessment of drug coating stability within the stent using scanning electron microscopy (SEM); 2) determination of ciprofloxacin and azithromycin release kinetics; and 3) assessment of anti-biofilm activities against Pseudomonas aeruginosa.

Results:

The ciprofloxacin nanoparticle-suspension in the inner layer was confirmed by zeta potential. Both ciprofloxacin (60 μg) and azithromycin (3mg) were uniformly coated on the surface of the PLLA stents. The CASS showed ciprofloxacin/azithromycin sustained release patterns, with 80.55 +/− 11.61 % of ciprofloxacin and 93.85 +/− 6.9 % of azithromycin released by 28 days. The CASS also significantly reduced P. aeruginosa biofilm mass compared to bare stents and controls (RODUs at OD590, CASS= 0.037 +/− 0.006, bare stent = 0.911 +/− 0.015, and control = 1.000 +/− 0.000, p < 0.001, n = 3).

Conclusions:

The CASS maintains a uniform coating and sustained delivery of ciprofloxacin and azithromycin, providing anti-biofilm activities against P. aeruginosa. Further studies evaluating the efficacy of CASS in a preclinical model are planned.

Keywords: Pseudomonas Aeruginosa, Ciprofloxacin, Azithromycin, Sinus Stent Biofilm, Chronic Rhinosinusitis

INTRODUCTION

Chronic rhinosinusitis (CRS) is a common inflammatory disease of the nose and paranasal sinuses characterized by persistent inflammation and bacterial infection of the mucosal surface.1,2 Bacterial biofilms produced by certain pathogens such as Pseudomonas aeruginosa reduce antibiotic penetration and lead to interventional failure in recalcitrant CRS.3–5 Therapeutic approaches that deliver large antibiotic concentrations at infected sites for an extended period of time would effectively eradicate the bacteria protected in biofilms, but avoid adverse effects associated with long-term systemic antibiotic treatment.6

The drug eluting sinus stent is a recently introduced method of delivering therapeutic agents directly into inflamed sinuses in a sustained manner, while mitigating the safety concerns regarding drug side effects.7 Using a ciprofloxacin (broad spectrum fluoroquinolone antibiotic)-eluting sinus stent, our recent studies demonstrated that a significant biofilm reduction of P. aeruginosa strains resulted in clearance of acute in-vivo rabbit sinusitis.8–11 However, the hydrophilic character of active ciprofloxacin (ciprofloxacin HCl) limits its topical use in a single layer coated sinus stent due to its initial burst release of ciprofloxacin.12,13 To create prolonged release without an initial burst, our previous study suggested the use of dual coated sinus stents, where ciprofloxacin (encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles) was coated in the inner layer and a hydrophobic drug was coated in the outer layer of the stent.14 In that study, ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, was chosen as a model drug to coat the hydrophobic outer layer to improve nasal and sinus mucociliary clearance (MCC) while providing synergistic anti-biofilm activity against P. aeruginosa.14,15 Despite promising outcomes of the co-delivery of the two drugs in a sustained continual release, ivacaftor is not readily available in a clinical setting to treat non-cystic fibrosis CRS, as it has FDA approval only for select CFTR mutations in CF.16

Azithromycin is a macrolide antibiotic with hydrophobic properties that has both anti-inflammatory and antibacterial activity.17,18 In the current study, azithromycin was explored as a therapeutic for the outer layer coating of the sinus stents to avoid burst release of hydrophilic ciprofloxacin. Incorporating azithromycin and ciprofloxacin together in a sinus stent could deliver a sustained prolonged release of ciprofloxacin while providing further antibacterial as well as anti-inflammatory effects to treat chronic inflammation in CRS by azithromycin.18 The objectives of this study were to fabricate a ciprofloxacin-azithromycin sinus stent (CASS) and to assess pharmacodynamic properties and efficacy in vitro.

MATERIALS AND METHODS

1. Materials

Azithromycin was purchased from TCI America (Portland, OR). Ciprofloxacin HCl (99.5% purity) was obtained from GenHunter Corporation (Nashville, TN). All other chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO).

2. Fabrication of a ciprofloxacin-azithromycin sinus stent (CASS) for in vitro analysis

Ciprofloxacin-nanoparticle suspension for the inner layer coating of the CASS

To create a coating solution containing ciprofloxacin for the inner layer, a nano-precipitation method was developed. First, the aqueous ciprofloxacin solution was prepared by dissolving 40 mg of ciprofloxacin into 1.5 ml of deionized water. Separately, Eudragit RS 100 (a copolymer of ethyl acrylate, methyl methacrylate, and a low content of methacrylic acid ester with quaternary ammonium groups) were dissolved into acetone to prepare a 35% Eudragit RS 100 solution. Then 1.5 ml of the aqueous ciprofloxacin solution and 1.5 ml of 35% Eudragit RS 100 solution were mixed and sonicated for 30 minutes to obtain a ciprofloxacin-nanoparticle suspension. Eudragit RS 100 has commonly been used in sustained-release pharmaceutical formulations to encourage a longer lasting effect.19

Azithromycin polymeric solution for the outer layer coating of the CASS

The outer layer coating solution was composed of an acrylate/ammonium methacrylate copolymer (Eudragit RL 100, Evonik) and azithromycin, which is a hydrophobic and ethanol-soluble molecule. To create an outer coating solution, 40 mg of azithromycin was dissolved into 1.5 ml of absolute alcohol and mixed with 1.5 ml of the 35% acrylates/ammonium methacrylate copolymer solution.

Coating ciprofloxacin-nanoparticle suspension and azithromycin solution onto biodegradable poly-D/L-lactic acid (PLLA) stents

To create the CASS, model biodegradable poly-D/L-lactic acid (PLLA) stents (Biogeneral, Inc., San Diego CA) were utilized in this study. Dual coating layers were fashioned onto the PLLA stents. First, the inner layer was coated with the ciprofloxacin-nanoparticle suspension. The stents were completely dried and placed in a vacuum for further coating processing. Next, the azithromycin-containing solution was used to create the outer layer. Coated CASSs were subject to an additional drying process in a vacuum for 2 days at room temperature. Sixty μg of ciprofloxacin was coated in the inner layer, while 3 mg of azithromycin was incorporated into the final CASS.

3. In vitro release profile of the CASS

For assessing the ciprofloxacin and azithromycin release kinetics in the CASS, two different groups of stents were prepared: 1) single ciprofloxacin coated stents and 2) dual coated stents containing ciprofloxacin in the inner layer and azithromycin in the outer layer. All of the samples (n=3 in each group) were incubated in 4 mL of sterilized phosphate buffered saline (PBS) at 37 °C for up to 28 days, and were subject to a periodic collection. To measure the released ciprofloxacin concentration, a ciprofloxacin enzyme-linked immunosorbent assay (ELISA) kit (REAGEN™, Moorestown NJ) was used according to the manufacture’s protocol. The azithromycin concentration was assessed by a spectrophotometric method, as described previously, with a slight modification. This protocol was based on the reduction of potassium permanganate in alkaline solution in the presence of azithromycin.20 200 μl of potassium permanganate (0.012M) solution and 200 μl of potassium carbonate (0.1M) solution were mixed, and subsequently 200 μl of a collected sample was added. Deionized water was added to make 2 ml of final solution and mixed thoroughly. The absorbance of samples was measured at 547 nm using a microplate reader (Synergy HK, BIO-TEK Instruments, Winooski, VT).

4. Evaluation of anti-biofilm activity of CASS

Quantitative analysis by crystal violet staining

Based on previous work, a crystal violet assay was used to assess the efficacy of the CASS against P. aeruginosa (PAO-1 strain) biofilms.21 Stents loaded with drugs were placed in a 48-well tissue culture plate. The stents were placed into Luria-Bertani (LB) media and then inoculated with 1×106 PAO-1. Stents without loaded drugs (bare stents) served as negative controls. After 3 days, the attached biofilm was assessed as previously described.21 900 μL of 0.1% (w/v) crystal violet was used to stain the biofilms. Next, 900 μL of 30% acetic acid was used to dissolve the PAO-1 biofilms and release the conjugated crystal violet dye. Absorbance was measured at 590nm to quantify the amount of crystal violet present.

Quantitative analysis by confocal laser scanning microscopy (CLSM)

To create pre-formed PAO-1 biofilms, PAO-1 was cultured for 24 hours on 14mm glass coverslips within a 35mm dish (MatTek, Ashland, MA). In the pre-formed PAO-1 biofilms, stents containing loaded drugs (ciprofloxacin and azithromycin) were placed in a 24-well tissue culture plate and cultured for an additional 3 days. Stents without loaded drugs (bare stents) were also introduced to serve as a negative control. To visualize both viable and dead bacterial populations, biofilms were stained with SYTO9 and propidium iodide (PI) (BacLight™ Live/Dead Bacterial Viability Kit; Molecular Probes, Eugene, OR). The biofilm was three-dimensionally reconstructed using NIS Elements microscopy imaging software and quantified. The proportions of live and dead bacteria were also quantified using BioFilmAnalyzer v.1.0 (https://bitbucket.org/rogex/biofilmanalyzer/downloads/)22 by counting fluorescence specific pixels in digital fluorescent images. Four different images per condition were selected for analysis.

5. Statistical Analysis

All experiments were performed in triplicate. Statistical analysis was performed with GraphPad Prism 6.0 (La Jolla, Ca). For assessing the anti-biofilm activity of stents, a one-way ANOVA was performed with a post-hoc Dunnett’s multiple comparison test. Significance was set at p < 0.05. Normalized values for relative biofilm quantification were expressed as ± standard error of the mean. For analyzing the difference between control and stents in the CLSM, t-tests were performed.

RESULTS

Structural morphology of the CASS

To examine the dual coated structure of the proposed CASS, a cross-sectional view of the CASS was imaged using scanning electron microscopy (SEM) (Figure 1). The ciprofloxacin-nanoparticle suspension was initially embedded within an acrylate and ammonium methacrylate copolymer polymeric matrix to create the inner layer on the PLLA stent surface (Figure 1A). When characterized by a zeta potential, the ciprofloxacin-nanoparticle suspensions were measured as +45.27 +/− 0.87 mV. Since ciprofloxacin is a negatively charged compound, the overall positive zeta-potential values demonstrated that the ciprofloxacin was encapsulated within the positively charged acrylates/ammonium methacrylate copolymer. Using an image analysis of SEM, the average thickness of the inner layer was observed as 120.9 +/− 4.9 μm. The average thickness of the outer layer was 256.2 +/− 14.60 μm, which was about twice of that of the inner layer (Figure 1B). Cross sectional images (Figure 1B) of the CASS demonstrates that the outer layers can be distinguished from the inner layer.

Figure 1.

Figure 1.

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Scanning electron microscopy (SEM) images of the ciprofloxacin and azithromycin sinus stent (CASS). The scale bar is located at lower right. * : ciprofloxacin (inner) layer, **: azithromycin (outer) layer.

A. Cross sectional view of the single layer of ciprofloxacin

B. Cross sectional view of the dual layers of ciprofloxacin and azithromycin

In vitro release profile of the CASS

To demonstrate the ability of the dual coated stent to provide sustained release of ciprofloxacin, the release kinetics of ciprofloxacin (60 μg) and azithromycin (3mg) from the CASS group was compared to that of a single coated ciprofloxacin stent (Figure 2A). Single coated stents with ciprofloxacin only exhibited a burst release pattern over 10 days. 60.16 +/− 14.65 % of coated ciprofloxacin was released by 2 days, and 83.81 +/− 7.51 % by 5 days. At 10 days, nearly 100% of the coated ciprofloxacin was eluted. In contrast, the dual coated CASS group demonstrated a sustained release of ciprofloxacin over a 28-day period. Briefly, 25.84 +/− 8.47 % of coated ciprofloxacin in the inner layer was released by 10 days, and 65.11 +/− 12.05 % by 21 days. At 28 days, most of the drug was released (80.55 +/− 11.61 %). Azithromycin also had sustained release throughout the study as follows: week 1 = 0.064 ± 0.061 mg/day, week 2 = 0.173 ± 0.026 mg/day, week 3 = 0.132 ± 0.012 mg/day and week 4= 0.063 ± 0.015 mg/day (Figure 2B).

Figure 2.

Figure 2.

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In vitro release profile of ciprofloxacin and azithromycin for 28 days. A) ciprofloxacin releasing profiles from single coated ciprofloxacin stents (open circle, n=3) and dual coated ciprofloxacin-azithromycin stents (closed square, n=3). B) azithromycin releasing profile from the CASSs (close circle, n=3)

CASS: ciprofloxacin-azithromycin sinus stent

Evaluation of anti-biofilm activity of the CASS against P. aeruginosa biofilms

Crystal violet staining

A standard crystal biofilm assay was used to measure the anti-biofilm activities of CASS. To determine the inhibition of P. aeruginosa PAO-1 biofilms by the CASS, 4 conditions were studied: 1) CASS, 2) bare stent (which is a PLLA stent without coating), and 3) control (Figure 3). After inoculating 1×106 PAO-1 in each condition, P. aeruginosa PAO-1 biofilms were developed for 72 hours and then subjected to crystal violet staining. The CASS significantly decreased P. aeruginosa PAO-1 biofilm mass compared to other conditions. Relative biofilm values calculated by relative optical density units (RODUs) were CASS = 0.037 +/− 0.006, bare stent = 0.911 +/− 0.015, control = 1.000 +/− 0.000 (p < 0.001, n = 3).

Figure 3.

Figure 3.

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Effect of CASS on the inhibition of Pseudomonas aeruginosa PAO-1 biofilm formation. CASS markedly inhibited the final P. aeruginosa PAO-1 biofilm mass. *** and **** indicate statistical significance when compared to control (p < 0.001 and p < 0.0001, n=3, respectively).

To evaluate the eradication of PAO-1 biofilm by CASS, PAO-1 biofilms were made by inoculating the 1×106 PAO-1 into LB media and cultivating them for 24 hours. 3 groups were subject to the following experiments. Samples were placed into the preformed PAO-1 biofilms and cultured for an additional 3 days. The 3 groups were 1) CASS, 2) bare stent (which is a PLLA stent without coating), and 3) control (Figure 4). Relative biofilm values compared to control at OD590 were CASS = 0.463 +/− 0.183, bare stent = 0.964 +/− 0.209, and control = 1.000 +/− 0.000 (p < 0.01, n = 3, respectively). As expected, the CASS group exhibited a significant reduction in biofilm mass compared to controls.

Figure 4.

Figure 4.

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Effect of CASS on the eradication of preformed Pseudomonas aeruginosa PAO-1 biofilms (1 day). CASS markedly reduced the final P. aeruginosa PAO-1 biofilm mass. * and ** indicate statistical significance when compared to control (p < 0.05 and p < 0.01, n=3, respectively).

CLSM analysis

The CASS were placed in the P. aeruginosa inoculated media for 1 day to demonstrate their efficacy against PAO-1 biofilms (Figure 5). When cultured with the CASS, the thickness of biofilms was significantly reduced; 21.60 +/− 3.94 μm, as compared to 29.63 +/− 1.47 μm in the controls (p = 0.0062), indicating that the CASS stent successfully inhibited their formation. Based on image analysis, the percentage of living PAO-1 cells in the CASS was significantly decreased (% of live cells; control without CASS = 94.19 +/− 5.37 % and CASS = 9.86 +/− 3.95 %; n = 3 per condition, p < 0.0001) (Figure 6C).

Figure 5.

Figure 5.

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Effect of the CASS on Pseudomonas aeruginosa PAO-1 biofilm formation. Representative confocal laser scanning microscopy (CLSM) images of PAO-1 biofilms with and without CASS after 24 hours. The maxium intensity projection images were used to create each panel. A plane view (square) shows the biofilm while the right and bottom images displays the side view of the biofilm. The scale bar indicates 50 μm.

A: PAO-1 biofilm with bare stent

B: PAO-1 biofilm with CASS

C: % of Live Cells between Control and CASS

Figure 6.

Figure 6.

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The efficacy of CASS on the eradication of preformed Pseudomonas aeruginosa PAO-1 biofilms. Representative confocal laser scanning microscopy (CLSM) images of PAO-1 biofilms were captured after a 3 day cultivation period with or without placing CASS to assess its effect on preformed PAO-1 biofilms. The maxium intensity projection images were used to create each panel. A plane view (square) shows the biofilm while the right and bottom images displays the side view of the biofilm. The scale bar indicates 50 μm.

A: PAO-1 biofilm with bare stent

B: PAO-1 biofilm with CASS

C: % of Live Cells between Control and CASS

To assess the ability of the CASS to eradicate preformed biofilms, CASS were placed on 1-day old PAO-1 biofilms and cultured for additional 3 days (Figure 6). There was a marked reduction in living PAO-1 cells in the biofilm mass in the presence of CASS. After 4 days, the percentage of living cells with CASS was 00.00 +/− 00.00 % whereas control without CASS represented 66.19 +/− 6.73 %, of living PAO-1 cells (p < 0.0001, n = 3). In addition, P. aeruginosa PAO-1 biofilm mass was significantly reduced by the CASS stents. The PAO-1 biofilm height with CASS (14.7 +/− 0.76 μm) was markedly lower than those from controls (44.68 +/− 5.24 μm) (p = 0.001). However, it should be noted that the control had a significant number of dead cells because of nutrient deprivation during the 4-day period of cultivation.

DISCUSSION

CRS is a chronic inflammatory disease that is strongly associated with recalcitrant bacterial infections such as P. aeruginosa.23 Bacterial biofilms stimulate innate immune reactions in the nasal epithelium, thus contributing to the severity of CRS.24 Anti-inflammatory agents aid efficacious management of CRS.18,25 Azithromycin has well described anti-inflammatory properties which can be attributed, at least partially, to inhibition of IL-8 production by macrophages, along with weak bactericidal effects.26–29 Long term, low dose azithromycin has been utilized for chronic inflammatory diseases, including CRS.30 The combination of antibiotics and immune-modulatory agents has immense potential to treat recalcitrant CRS.

Our previous studies used the CFTR potentiator, ivacaftor, to control the release of hydrophilic ciprofloxacin due to its hydrophobic properties and its role as a mucociliary activator.14,31 In the current study, we successfully coated PLLA stents with a dual layer of ciprofloxacin (inner layer) and azithromycin (outer layer) on a PLLA stent according to zeta potential and observable differentiation between layers on SEM. Azithromycin has hydrophobic characteristics similar to ivacaftor, but already has FDA approved indications for sinusitis, low cost, and anti-inflammatory properties.29 The CASS was able to control the release of ciprofloxacin over 28 days. The initial amount of azithromycin loaded in the CASS was 3 mg. Although the CASS released azithromycin well below the minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa PAO-1 (> 128 μg/ml)32, the drug was utilized in this study only for its hydrophobic properties and anti-inflammatory effects. Based on the pharmacokinetic analysis of azithromycin in patients given oral doses (500mg/day or 1g/day), the maximal concentrations of azithromycin in bronchial alveolar lavage fluid (BAL) were between 0.72 +/− 0.06 μg/ml (500mg) and 1.41+/− 0.06 μg/ml (1g) in BAL in human samples after 12 hours.33 Considering the total amount of nasal mucus production per day in humans (1~2L/day)34, ideal nasal mucus concentration of azithromycin in each nose should be approximately 1.5 mg/day (1.5 μg/ml x 1L). Therefore, based on our data (week 2 = 0.173 ± 0.026 mg/day, week 3 = 0.132 ± 0.012 mg/day), we should use a CASS stent with 10x higher surface area to reach this concentration in human clinical trials. The anti-inflammatory effects of the azithromycin should provide additional benefit for eventual use in preclinical animal models and human studies.

Activity of the CASS against the PAO-1 strain of P. aeruginosa was also investigated in the current study. The ratio of the area under the curve (AUC) against minimum inhibitory concentration (MIC) of ciprofloxacin was calculated from our data: approximately 25.83 at week 1 (day 7) and 41.33 at week 3. The MIC of P. aeruginosa PAO1 strain H103 is 0.06 μg/ml35. These results indicate the CASS released an effective concentration of ciprofloxacin over 28 days, thereby achieving a long-term antibiotic delivery method to treat a P. aeruginosa infection. Indeed, robust reduction of biofilm mass was noted with the CASS by both crystal violet staining (p<0.01) and imaging analysis with confocal microscopy (p<0.0001). In future in vivo evaluation in animal models, we will incorporate a 10-fold higher concentration of both ciprofloxacin and azithromycin to compensate for 10-fold increase in surface area compared to the current model of CASS. The concentration of ciprofloxacin (600 μg) used in vivo will be sufficient to reach the minimum goal of the AUC0–24/MIC values for ciprofloxacin.36,37 Hence, we postulate the CASS therapeutic strategy would also successfully deliver ciprofloxacin in an extended manner in vivo.

There are several limitations to this study. While we have created a dual coating stent (hydrophilic and hydrophobic layers) with controlled release of two drugs, creating a single drug coated control stent with similar drug release kinetics (controlled release) is technically difficult because of the initial burst release of single drug and thus may not represent a true control. Additionally, only 1 strain of Pseudomonas was tested in this study. This bacteria is well known for its intrinsic resistance to a variety of antimicrobial agents and toxic compounds and there is significant variability among clinical isolates.38 Further studies are planned to test the efficacy of this methodology against many multi-drug resistant strains of Pseudomonas. Additionally, the drug release with the current stent will need modifications to improve concentrations on future iterations prior to planned animal studies.

CONCLUSION

The CASS was successfully developed by incorporating ciprofloxacin and azithromycin into two different layers. The dual layered stent demonstrated an extended release of both azithromycin and ciprofloxacin over 28 days. Additionally, the CASS not only decreased the formation of P. aeruginosa PAO-1 biofilms, but also the eradicated preformed biofilms. The novel double layered sinus stent may provide therapeutic advantages over current treatment strategies for recalcitrant bacterial infections in CRS. Future studies assessing the CASS stents in preclinical animal models with active maxillary P. aeruginosa sinusitis are planned.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1 R01 HL133006-03) and National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482-05, CF Research Center Pilot Award) to B.A.W. and NIH/National Institutes of Allergy and Infectious disease (K08AI146220), John W. Kirklin Research and Education Foundation Fellowship Award, UAB Faculty Development Research Award, American Rhinologic Society New Investigator Award, and Cystic Fibrosis Foundation Research Development Pilot grant (ROWE15R0) to D.Y.C.

Footnotes

Manuscript was presented at the American Rhinologic Society Meetings, New Orleans, LA, Sep 13th, 2019.

Bradford A. Woodworth, M.D. is a consultant for Olympus and Cook Medical. The authors whose names are listed other than Bradford A. Woodworth, M.D. certify that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.

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