The cornea is a transparent layer that covers the front of the eye and plays a vital role in protection against several abiotic like fine hairs and sand, and biotic factors such as bacteria, fungi and virus . Ocular fungal infection is a microbial disease caused by a range of fungi such as Candida, Aspergillus and Fusarium species . According to a recent publication, around 1 million fungal keratitis cases have been estimated per year . Treatment of fungal infection is challenging because of anterior ocular barriers like corneal epithelium and tear fluid, limiting drug availability and the lack of efficient drug delivery systems . A limited number of antifungal drugs (azoles, polyenes and echinocandins) are currently used to treat fungal infection . However, both azoles and echinocandins groups develop drug resistance, but to a lower extent than polyene’s antifungal drugs such as Amphotericin B (AMP-B) .
AMP-B was discovered seven decades ago as a secondary metabolite from soil species of actinomycetes called Streptomyces nodosus and widely used to treat fungal infections either intravenously or topically [7–9]. The antifungal activity of AMP-B is attributed to its ability to obstruct the synthesis of the fungal cell wall by binding with sterols molecules forming ergosterol-AMP-B complex that leads to pore formation and leaking of intracellular ions which eventually causes fungal cell destruction and death .
The conventional formulation of AMP-B is AMP-B deoxycholate (Fungizone®), commonly administered as an injection and suffers from limited dose toxicity such as infusion-related reaction and nephrotoxicity, and this often prevents further administration leading to incomplete treatment course . Typical formulations such as eye drops of AMP-B provide inadequate drug absorption and poor corneal bioavailability . Ocular administration (subconjunctival injection) also showed some toxicity which was attributed to the presence of deoxycholate .
In an attempt to improve the therapeutic index of AMP-B, different lipid formulations were developed, such as AMP-B lipid complex (ABLC), liposomal AMP-B (L-AmB), and AMP-B colloidal dispersion (ABCD) . Although lipid formulations were shown as safer alternatives to conventional aqueous AMP-B formulations, localised administration of AMP-B lipid formulations was shown to induce toxicity due to the presence of phospholipids that cause pseudo-hyperphosphatemia .
Drug delivery systems using biodegradable polymers were emerged to increase solubility and reduce drug toxicity. Biodegradable polymers could potentially improve local residence time, prolong drug action and improve bioavailability [13–15]. Incorporation of biodegradable, biocompatible polymers including oxidized arabinogalactan, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), copolymer polylactic acid and polyglycolic acid (PLGA)-TPGS (tocopheryl polyethene glycol succinate) shown in vitro and in vivo decrease in toxicity of AMP-B [16, 17]. However, it was noticed that all previous studies were devoted to developing AMP-B formulations for systemic administration, using oral [18, 19] or intravenously [20, 21]. Limited studies demonstrated the use of topical AMP-B formulations with biodegradable polymers for ocular delivery. For instance, collagen was used as a shield of contact lenses for releasing AMP-B on the cornea’s surface . Furthermore, chitosan was incorporated in poly (lactic acid) to produce self-assembled nanoparticles for ophthalmic delivery of AMP-B .
Microneedles (MNs) have emerged as a practical approach for controlled drug release locally. This promising technique was successfully employed for improving drug delivery through the skin [25–28] and further developed as an efficient approach for ocular delivery by our group [30, 31]. MNs are minimally invasive micron-sized needles ranging from 60 µm to 2000 µm in height and varying arrays [32, 33], and economically cheaper than other delivery strategies. For example, the design and production of single biodegradable MN for medical use with AMP-B could be more affordable, whereas advanced liposomal formulations for AMP-B costs around $85 per 50 mg/vial .
We have previously demonstrated the use of dissolve PVP MNs to improve ocular drug delivery of large model molecules (fluorescein isothiocyanate-dextran) by disturbing the cornea's barrier function sclera [30, 31]. Similarly, recent studies by Bhatnagar et al. showed that delivery of besifloxacin loaded in PVP/PVA MNs successfully treated bacterial keratitis . Also, Sachan et al. demonstrated that polyglycolic acid MNs for transdermal delivery of AMP-B . Roy et al. fabricated liposomal AMP-B to load into the polymeric MNs . However, liposomal formulation reduced the loading capacity in micron size MN tips, and high loading of lipidic liposomal formulation could affect the mechanical strength of MN adversely. Additionally, increasing MN arrays or patch size for the corneal application can cause corneal scarring. Therefore, this study aims to investigate to directly load AMP-B in biodegradable PVP/PVA and hyaluronic acid (HA) based rapidly dissolved polymeric MNs to treat ocular fungal infection via intracorneal route. The MNs were thoroughly characterized for their mechanical strength, dissolution characteristics, insertion depths in the artificial membrane and corneal tissues. Multiphoton microscopic studies were conducted to investigate the depth of AMP-B penetration and distribution within the corneal tissues. In vitro antifungal activity of AMP-B encapsulated MNs and biocompatibility of the polymers was also investigated.