Chitosan-Based Crosslinking for Controlled Topical Drug Release in Rhinosinusitis

INTRODUCTION

Chronic rhinosinusitis (CRS) is defined as a persistent inflammation in the sinonasal cavities triggered by multifactorial causes such as genetic, environmental, and infectious factors.^1,2 Therapeutics, such as corticosteroids and antibiotics, have been widely studied to address the potential for prolonged topical drug delivery, thereby providing positive clinical outcomes in CRS without systemic side effects through oral intake.^3,4 From our previous in vitro release assays, the water solubility of the drug itself could affect the sustained release profile of the therapeutics: hydrophilic drugs exhibited improved control over their release when anchored or encapsulated by a vehicle or another hydrophobic drug.^5,6 Chitosan, a natural polysaccharide obtained mainly from marine crustaceans, is a promising drug delivery vector for therapeutics owing to its biocompatibility and low toxicity.⁷ This study aimed to characterize the release profiles of ciprofloxacin (hydrophilic) and triamcinolone acetonide (TA, hydrophobic) when crosslinked with chitosan-based on their water solubility.

METHODS

TA injectable suspension (40mg/ml) (Kenalog^®-40, Bridgewater, NJ) and ciprofloxacin HCl with a purity of 99.5% (GenHunter, Nashville, TN) were used in this study. All other chemicals and reagents (chitosan low molecular weight, glutaraldehyde (50% (w/w) aqueous solution), _DL-glyceraldehyde) were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Hampton, NH). 0.05% chitosan microsphere particles were prepared based on a study by Gaspar et al. for the development of levofloxacin-loaded microspheres for dry aerosol therapy.⁸ Ciprofloxacin (hydrophilic) and TA (hydrophobic) were crosslinked with 0.05% chitosan microsphere. Then, to mimic the topical placement of therapeutics on sinonasal mucosa, punch biopsies (4 mm) of ovine forestomach matrix (Myriad Matrix^™, New Zealand) on transwell filters were impregnated with ciprofloxacin (0.2 mg) or TA (1.0 mg), stored in a 37 °C incubator. At 6hrs, 1, 2, 3, 5, 7, and 10 days, the saline solution underneath the filter was collected and replaced with an equal volume of saline. TA and ciprofloxacin concentrations from each solution were measured using an enzyme-linked immunosorbent assay kit (TA: Neogen^Corp, Lansing, MI; Ciprofloxacin: Abcam Limited, United Kingdom) according to the manufacturer’s protocol. All experiments were performed in triplicate. Analysis was performed with GraphPad Prism 6.0 (La Jolla, Ca). Each value was represented as mean +/− standard error of the mean.

RESULTS

We successfully generated therapeutics (ciprofloxacin and TA) crosslinked with chitosan using an optimal chitosan concentration (0.05%) and an ideal ratio between therapeutics and chitosan (1:4) (see Supplement). This formulation allowed to study the prolonged release of therapeutics in this study.

There was no difference in immediate release profiles (initial burst release) between chitosan-crosslinked ciprofloxacin and ciprofloxacin alone at 6hrs (Fig 1A). However, once crosslinked, the chitosan could hold 40% of the loaded ciprofloxacin, and the release of hydrophilic ciprofloxacin (80%) was delayed for up to 3 days. The statistical difference (p<0.05) of released ciprofloxacin between the two groups was noticed only at day 1. On the contrary, no initial burst release was seen in chitosan-crosslinked TA at 6hrs (Fig 1B). 95% of chitosan-crosslinked TA was released up to day 7, while 95.7% of TA alone was released by day 3. The two groups’ statistical differences of released TA were noticed at multiple time points (6hrs, days 1/2/3).

Figure 1.

In vitro release profiles of Ciprofloxacin (A) and TA (B).

A: Once crosslinked, the chitosan could hold 40% of the loaded ciprofloxacin, and the release of hydrophilic ciprofloxacin (80%) was delayed for up to 3 days. (* p<0.05)

B: 95% of chitosan-crosslinked TA was released up to day 7, while 95.7% of TA alone was released by day 3 (chitosan-crosslinked TA 94.8 +/− 8.7% on day 7 vs. TA alone 95.7 +/− 5.9% on day 3). (* p<0.05, ** p<0.01)

DISCUSSION

The initial therapeutic approaches of CRS target reducing inflammation of the mucosal lining, controlling bacterial colonization, and managing the host response to environmental triggers.⁹ A number of studies focusing on topical treatments have garnered interest as a means to improve the management of CRS.³ Locally delivered therapeutics can directly act on the inflamed sinonasal mucosa, providing a higher concentration while evading systemic adverse reactions.¹⁰ For sustained sinonasal drug delivery, vehicles are necessary to keep active therapeutics in the sinonasal cavity and minimize a burst release into the mucus layer during mucociliary clearance (MCC). Of those multiple candidate vehicles, chitosan was selected based on the following characteristics: 1) ability to adhere to extensive mucosal surface areas, 2) protecting therapeutic particles from MCC, 3) easiness in combining with other biomaterials, and 4) non-toxic/anti-inflammatory/antibacterial characteristics.⁷

Our findings demonstrated that once crosslinked with chitosan, prolonged drug release was observed regardless of water solubility. Crosslinking held about 40% of loaded ciprofloxacin, twice longer than non-crosslinked ciprofloxacin before its release. However, hydrophilic ciprofloxacin was released relatively quickly, requiring repeated administration compared to the hydrophobic TA. Similar results were demonstrated by Gaspar et al. regarding the levofloxacin-loaded chitosan microsphere with negligible or minimal difference between crosslinked and non-crosslinked systems.⁸ The hydrophilic drugs may require different strategies (e.g., encapsulation) to obtain a sustained release profile. Our in vitro study could guide applying off-label topical drugs with chitosan-based dressing biomaterials in CRS.

This study possesses several limitations. The primary constraint of this research lies in the fact that the release of ciprofloxacin and TA in vitro may not entirely reflect their performance in vivo, including the bioavailability of ciprofloxacin after crosslinking. Furthermore, the dissolution patterns of biomaterials within the human body could vary based on local environmental factors such as humidity, pH, temperature, or immune responses near the placement site. Despite conducting our experiments under controlled conditions mimicking body temperature and moisture, these in vivo complexities were not fully accounted for. Lastly, additional clinical benefits of chitosan were not addressed in this in vitro assay, which could be significant. Even though some studies have indicated the antibacterial efficacy of chitosan at higher concentrations (0.0125% or 0.05% (which aligns with our concentration)), the antimicrobial activity of chitosan against Pseudomonas species remains a topic of debate.⁸

KEY POINTS.

• Chitosan is a promising drug delivery vector for therapeutics owing to its biocompatibility.

• Once crosslinked with chitosan, prolonged drug release was noted regardless of hydrophilicity.

• The hydrophilic drugs may require different strategies to obtain a sustained release profile.