**3. Alternative treatment approaches in bacterial keratitis novel drug delivery methods**

Antibiotic eye drops were the most common first-line treatment option and this requires high drug compliance for the therapeutic outcome. The frequent eye drops administration makes them wearisome and thus poor healing.

Exploiting contact lenses for constant drug delivery (zero-order kinetics) was highly challenging. The soft contact lenses exhibiting the feature of drug uptake and release were explored to extend their use in attaining therapeutic index. The pharmacokinetic profile in contact lens drug release is nonlinear kinetics, as there is an immediate drug release and later it tends to decrease to a sub-therapeutic level in the subsequent hours.

Research has also focused on the controlled release of medications from delivery systems incorporated into a contact lens hydrogel material, including copolymerizing the hydrogel and poly (hydroxyethyl methacrylate) (pHEMA) with other monomers.

Hydrogel prototype lenses are used to release the drug in the form of microemulsions. Only first-order kinetics was achieved by manipulating the surface of the contact lenses with the drug-containing liposomes. The physiochemical environment of the human eye with alkaline pH and physiological temperature were the odd factors that prohibit the sustained release of the drug.

Formerly many non-contact lens techniques were attempted in futile to achieve long-term drug release. Ocusert by Alza Corp., Palo Alto, CA, was specifically designed to be placed in the cul-de-sac and had demonstrated zero-order kinetics and it was not widely used except to treat glaucoma, whereas the collagen shields require surgical removal of corneal epithelium to promote corneal re-epithelialization and thereby antibiotic prophylaxis. This method was also not being used due to its hostile natures such as difficulty in self-insert, requiring topical anesthesia, and replacement of a new collagen shield, every 3 days.

As a result, novel drug delivery methods are needed to increase compliance and therefore the efficacy of treatment.

The challenges in achieving the desired sustained release system in an ophthalmic drug were the bioavailability of the drug, the biocompatibility of the contact lenses, absorption and release of the drug in a zero-order kinetics to achieve an extendedrelease of the drug, etc. In the early 1960s, hydrogel contact lens were introduced and used as a drug-eluting contact lens and as a bandage contact lens. The hydrogel

contact lenses as bandage contact lenses were helpful in cornea protection and corneal re-epithelization with antibiotic drops. Unlikely the extended release of antibiotic eye drops could not be established in the hydrogel contact lenses.

A prototype contact lens for sustained drug delivery by incorporating a thin drug-PLGA [Poly (lactic-co-glycolic acid)] film into a pHEMA hydrogel [Poly (2-hydroxyethyl methacrylate)], and this polymer was used in the making of regular contact lenses also. This warrants a regular adjustment of polymer molecular mass and medication concentration in the drug-PLGA film, to reach the zero-order kinetics. This ocular drug delivery system was prominent to maintain therapeutic concentration for about a month. This prototype contact lens design was used as a platform for ocular drug delivery and therapeutic applications. The contact lenses are used as the antibacterial prototype lenses through antibiotic coating with ciprofloxacin-PLGA 65:35 films (pHEMA).

In this prototype contact lens phenomenon, there was an initial drug release in the first 24 hours, followed by this burst the prototype contact lens maintains zero-order kinetics for more than 4 weeks. For instance, 134 μg of ciprofloxacin per day was released constantly to maintain the zero-order kinetics. The ciprofloxacin (23%) was released from the lenses in a month.

A drug-eluting contact lens with a combination of drugs say, moxifloxacin (MF) and dexamethasone (DM), were experimented with. In this study, a polymeric contact lens using chitosan, glycerol, and polyethylene glycol (PEG) was developed along with MF and DM. Drug-loaded contact lenses were tested with a combination of drugs as well as individually, and all three lenses were compared to treatment with individual drug solutions. Both required therapeutic concentration and corneal drug distribution of MF were significant in drug-loaded contact lenses when compared to topically given drug solutions in rabbits and humans. It also features *in vitro* and *in vivo* antimicrobial activity through mucoadhesion by contact lenses [10].

The moxifloxacin in nanoparticles increased the corneal penetration compared to MF in solution. The improved therapeutic effect was obtained when *in situ* gel formation was combined with nanoparticles that is, nanoparticles can also be used to load antibiotics; moxifloxacin nanoparticles show increased corneal penetration. When the liquid gets into contact with the corneal surface, it forms an *in situ* gel that maintains bioavailability [11, 12].

Another breakthrough in nanoparticle research is molecular imprinting. Antibodies were formed through the conversion of nanoparticles into synthetic antibodies equivalent. These antibodies target the lipopolysaccharides in *P. aeruginosa*, in a keratitis model. Methicillin-resistant *Staphylococcus aureus* (MRSA) was also targeted in a similar approach.

Apart from lenses offering the sustained release of drugs, antimicrobial compounds have been incorporated into the lens itself; AGMNA, a metal–organic framework featuring silver (a natural antimicrobial agent), has been developed both for inclusion into the contact lens structure and as a lens disinfecting agent, with high effectiveness and minimal toxicity [13].

In the above study, the Metal–Organic Framework (MOF) of formula {[Ag6(μ3- HMNA)4(μ3-MNA)2]2 − ·[(Et3NH)+]2·(DMSO)2·(H2O)} (AGMNA), a known efficient antimicrobial compound which contains the anti-metabolite, 2-thionicotinic acid (H2MNA), was incorporated in polymer hydrogels using hydroxyethylmethacrylate (HEMA).

pHEMA@AGMNA-1 has antimicrobial activity against the microbial keratitis etiologies gram-negative *P. aeruginosa* and gram-positive *Staphylococcus epidermidis* and *S. aureus*. The following organism is incubated with pHEMA@AGMNA-1 discs with % bacterial viability say *P. aeruginosa*, *S. aureus*, and *S. epidermidis* [13]. Furthermore, pHEMA@AGMNA-1 exhibits low toxicity.

#### **3.1 Microemulsions**

Microemulsions are another novel method of ocular drug delivery and have shown a promising result in a combined *in vivo* and *in vitro* study [14]. A tiny droplet with a diameter of 10 to 100 nm is formed by the drug with the surfactant. The lipid-waterlipid sandwich of the cornea makes an effective microemulsions delivery [15]. The outer layer of the cornea is a barrier to hydrophilic substances but is lipid-soluble; thus, microemulsions can effectively deliver a drug to the stroma.

Antibiotics can also be similarly delivered to the eye by liposomes, a capsule made of a phospholipid bilayer. Furthermore, Mishra et al. found that contact lenses equipped with liposomes are capable of providing a stable release of antibiotics over 6 days, which was effective against *S. aureus in vitro*.

#### **3.2 Plasma and phage therapy**

Plasma and phage therapy was a novel therapeutic option in BK treatment. Plasma is an ionized gas capable of exhibiting antimicrobial properties *via* its ability to produce reactive oxygen species; it also exhibits wound healing and anti-inflammatory properties [16].

Reitberger et al. studied the argon-based plasma therapy and opined that it shall be successfully exploited in combination with antibiotics [16]. Phage therapy involves using a viral bacteriophage to infect and kill bacteria. There was only one study to support the efficiency of phage therapy against *P. aeruginosa* keratitis in mice [17]. Also, a case study reports the efficacy of phage therapy against MRSA keratitis. The effectiveness of phage therapy against a wide number of different non-ocular bacterial colonies has been confirmed by other studies, but there is a need for further investigation focusing specifically on *S. aureus* keratitis isolates.

#### **3.3 Photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL)**

It works on the mechanism of collagen fiber photopolymerization on the corneal tissue to get stiffened by applying a combination of ultraviolet A radiation and a chromophore (riboflavin). This is a non-invasive procedure performed with topical anesthesia.

The photoactivated chromophore and ultraviolet A light have antibacterial properties and are effective in treating infectious keratitis. The antibacterial mechanism involved here is inhibition of microbial replication, intercalation of the chromophore with microbial nucleic acids, RNA damage, DNA damage, cell wall damage, and oxidation of nucleic acid residues by reactive oxygen species, as well as increased resistance of the stiffened cornea to enzymatic damage from the microorganisms. Other potential advantages of UVA and riboflavin application over antibiotics include eliminating ocular surface toxicity and avoiding adherence issues associated with the need for frequent eye drop administration, among others [18].

PACK-CXL with ultraviolet A and riboflavin was applied on the day of diagnosis. According to the Dresden modified protocol, riboflavin 0.1% solution was administered to the cornea every minute for 15 minutes, followed by exposure to 370-nm UVA light

(with a fluence of 3 mW/cm2 ) from a distance of 1 cm for 30 minutes. Following this, the eye was given a saline rinse and a contact lens was placed. A post-operative regimen of 0.1% fluorometholone acetate eye drops was instilled for 2 days (4 times a day) and for 1 week (3 times a day). The contact lens was removed one day after placement.

The epithelial healing was monitored as a mark of recovery where the patient will receive antibiogram results based on topical antibiotic eye drops along with artificial tear eye drops. During this period, the patient will also wear UV protection glasses. The patient was observed for the presence or absence of corneal ulcer and a comparison was made for treatment response against different time points.

The significance of ulcer healing was moderate in the early weeks of the treatment i.e., from between Day 1 and Week 1. The healing tends to increase over time Month 3 > Month 1 > Week 1. Complete recovery in all treated eyes was accomplished except for four cases due to emergency surgery.
