Ophthalmic Drug Delivery

## 2nd Edition

Edited by Francisco Javier Otero-Espinar and Anxo Fernández Ferreiro

mdpi.com/journal/pharmaceutics

## **Ophthalmic Drug Delivery, 2nd Edition**

## **Ophthalmic Drug Delivery, 2nd Edition**

Editors

**Francisco Javier Otero-Espinar Anxo Fern´andez Ferreiro**

Basel • Beijing • Wuhan • Barcelona • Belgrade • Novi Sad • Cluj • Manchester

*Editors* Francisco Javier Otero-Espinar Department of Pharmacology, Pharmacy and Pharmaceutical Technology University of Santiago de Compostela (USC) Santiago de Compostela Spain

Anxo Fernandez Ferreiro ´ Hospital Pharmacy & Department and Pharmacology Group SERGAS and Health Research Institute Santiago Compostela Santiago de Compostela Spain

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Pharmaceutics* (ISSN 1999-4923) (available at: www.mdpi.com/journal/pharmaceutics/special issues/ophthalmic drugdelivery II).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Lastname, A.A.; Lastname, B.B. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-8799-8 (Hbk) ISBN 978-3-0365-8798-1 (PDF) doi.org/10.3390/books978-3-0365-8798-1**

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) license.

## **Contents**

Sustained-Release Microspheres of Rivoceranib for the Treatment of Subfoveal Choroidal Neovascularization

Reprinted from: *Pharmaceutics* **2021**, *13*, 1548, doi:10.3390/pharmaceutics13101548 . . . . . . . . **149**

## **Preface**

Research in ophthalmic drug delivery has developed significant advances in the few last years, and efforts have been made to develop more effective topical formulations to increase drug bioavailability, efficiency, and safety. Drug delivery to the posterior segment of the eye remains a great challenge in the pharmaceutical industry due to the complexity and particularity of the eye's anatomy and physiology. Some advances have been made with the purpose of maintaining constant drug levels in the site of action. The anatomical ocular barriers have a great impact on drug pharmacokinetics and, subsequently, on the pharmacological effect.

Despite the increasing interest in efficiently reaching the posterior segment of the eye with reduced adverse effects, there is still a need to expand the knowledge of ocular pharmacokinetics that allow the development of safer and more innovative drug delivery systems. These novel approaches may greatly improve the lives of patients with ocular pathologies.

> **Francisco Javier Otero-Espinar and Anxo Fern´andez Ferreiro** *Editors*

## *Article* **Topical Sustained Delivery of Miltefosine Via Drug-Eluting Contact Lenses to Treat Acanthamoeba Keratitis**

**Lin Chen 1,2,3,† , Liangju Kuang 3,\* ,† , Amy E. Ross 3 , Wissam Farhat 3 , Nikolay Boychev 3 , Sina Sharfi 3 , Levi N. Kanu 3 , Longqian Liu 1 , Daniel S. Kohane 4 and Joseph B. Ciolino 3,\***

**Abstract:** This study aimed to develop a miltefosine-eluting contact lens (MLF-CL) device that would allow sustained and localized miltefosine release for the treatment of Acanthamoeba keratitis. MLF-CLs were produced in three different miltefosine doses by solvent-casting a thin miltefosinepolymer film around the periphery of a methafilcon hydrogel, which was then lathed into a contact lens. During seven days of in vitro testing, all three formulations demonstrated sustained release from the lens at theoretically therapeutic levels. Based on the physicochemical characterization of MLF-CLs, MLF-CL's physical properties are not significantly different from commercial contact lenses in terms of light transmittance, water content and wettability. MLF-CLs possessed a slight reduction in compression modulus that was attributed to the inclusion of polymer-drug films but still remain within the optimal range of soft contact lenses. In cytotoxicity studies, MLF-CL indicated up to 91% viability, which decreased proportionally as miltefosine loading increased. A three-day biocompatibility test on New Zealand White rabbits revealed no impact of MLF-CLs on the corneal tissue. The MLF-CLs provided sustained in vitro release of miltefosine for a week while maintaining comparable physical features to a commercial contact lens. MLF-CL has a promising potential to be used as a successful treatment method for Acanthamoeba keratitis.

**Keywords:** acanthamoeba keratitis; controlled drug delivery; contact lens; miltefosine; PLGA

### **1. Introduction**

Acanthamoeba keratitis (AK) is a rare but potentially blinding corneal infection caused by free-living Acanthamoeba amoebae [1,2]. Inadequate disinfection of commercial contact lenses is one of the most common risks of AK [3–5]. With the increasing number of people wearing contact lenses, the prevalence of Acanthamoeba infections in the world is on the rise [6–10]. AK treatment at present is suboptimal and the prognosis of this condition is often poor [11]. First-line therapies for AK include topical polyhexamethylene biguanide (PHMB), propamidine isethionate, chlorhexidine, dibromopropamidine, and/or hexamidine [12,13], often requiring two or more drugs. However, the treatment duration is lengthy, complicated, and is often ultimately unsuccessful in complete eradication. Frequent eye drop instillation, as often as hourly throughout the day [14] is used in clinical practice, in part because of the limited bioavailability of topical eye drops [15]. Bioavailability from eye drops is estimated to be less than 5% because of the anatomic barriers in the anterior segment of the eye (e.g., tear film barrier and corneal barrier) [16,17]. In addition, trophozoites and cysts (two forms of Acanthamoeba's lifecycle) can remain in the cornea

**Citation:** Chen, L.; Kuang, L.; Ross, A.E.; Farhat, W.; Boychev, N.; Sharfi, S.; Kanu, L.N.; Liu, L.; Kohane, D.S.; Ciolino, J.B. Topical Sustained Delivery of Miltefosine Via Drug-Eluting Contact Lenses to Treat Acanthamoeba Keratitis. *Pharmaceutics* **2022**, *14*, 2750. https://doi.org/10.3390/ pharmaceutics14122750

Academic Editors: Francisco Javier Otero-Espinar and Anxo Fernández Ferreiro

Received: 12 October 2022 Accepted: 23 November 2022 Published: 8 December 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for over two years [2,18]. Non-adherence can lead to suboptimal dosing. In general, killing of amoebic trophozoites and cysts is a challenge.

Miltefosine is an alkylphosphocholine agent with a broad spectrum of antiparasitic properties, which has recently been used in AK. This drug is commonly used as an oral medication to treat leishmaniasis [19]. After obtaining orphan drug designation from the FDA in 2016, oral miltefosine has been shown to be effective in refractory AK in several recent case reports [20–23]. However, systemic administration can produce severe inflammatory responses [24,25]. Providing a topical treatment could mitigate or even eliminate miltefosine's systemic side effects. Although miltefosine eye drops were effective in the management of AK in hamster [26] and rat [27] models, a clinical study failed to replicate the results in the humans [28]. The ineffectiveness observed may be attributed to the low bioavailability of eye drops in general and the fact that all studied cases had advanced keratitis at the time of initiation of the topical miltefosine therapy.

Previously, we have described a contact lens drug delivery system involving the incorporation of a thin drug-poly[lactic-co-glycolic acid] (PLGA) film into a poly[hydroxyethyl methacrylate] (pHEMA) hydrogel. This system has previously been used to achieve zero-order release kinetics at therapeutically relevant concentrations with fluorescein and ciprofloxacin for up to one month [29].

In this study, we incorporated miltefosine into this contact lens system to create a therapeutic contact lens (MLF-CL) for the treatment of AK. Miltefosine was loaded at different concentrations and the physicomechanical properties (hydration, water contact angle, mechanical strength) of the MLF-CLs were investigated and compared with commercial contact lenses. Drug release kinetics were measured for a period of one week. In addition, the cytocompatibility and in vivo biocompatibility of the MLF-CL was evaluated.

#### **2. Materials and Methods**

### *2.1. Materials*

Miltefosine was purchased from Cayman Chemical (Ann Arbor, MI, USA). Liquid methafilcon and methafilcon contact lens blanks were purchased from Kontur Kontact Lens (Hercules, CA, USA). Poly(lactide-co-glycolide), 85% lactide, 15% glycolide (MW 190 KDa) was purchased from Merck and Cie (Schaffhausen, Switzerland). Hexafluoroisopropanol (HFIP) was purchased from Oakwood Chemical (Estill, NC, USA). Gibco phosphate-buffered saline (PBS), Dulbecco's Modification of Eagle's Medium, penicillinstreptomycin, and L-glutamine were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Biopsy punches were purchased from Sklar Instruments (Westchester, PA, USA). All other reagents were purchased from Millipore-Sigma (St. Louis, MO, USA), except where otherwise noted.

#### *2.2. MLF-CL Fabrication*

The drug-polymer solution was created by dissolving miltefosine and PLGA in HFIP, in ratios provided in Table 1. The polymer concentration was kept at 50 mg/mL. The film was created by using solvent casting. 50 µL of solution was then pipetted into a concavity lathed into a cylinder of dry polymerized methafilcon After 6 min of rotation on a spin coater (Model SC100, Best Tools LLC, St. Louis, MO, USA), the liquid HFIP evaporated, leaving a drug-polymer film. A 4 mm biopsy punch was used to define and remove the central diameter of the drug-polymer film to create a clear central aperture. The hydrogel blanks containing the films were placed on a desiccator under vacuum for two weeks to remove residual HFIP. The side of the film that was not yet in contact with methafilcon was then encapsulated in methafilcon through ultraviolet (UV) photopolymerization using ethylene glycol dimethacrylate (0.3%, crosslinker), Irgacure® 2959 (9 mg/mL, photoinitiator) and 3.5 min of UV light exposure at 120 mW/cm2 applied within a Zeta 7401 UV chamber (Loctite, Düsseldorf, Germany) for 3.5 min at 120 mW/cm2 . The methafilcon cylinder was then lathed into a contact lens that consisted of the drug-PLGA film fully encapsulated

within methafilcon, with the initial cylinder on the front curve and polymerized methafilcon on the back curve (Scheme 1). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 3 of 15

**Scheme 1.** Schematic illustration of the designed miltefosine-eluting contact lens (MLF-CL). **Scheme 1.** Schematic illustration of the designed miltefosine-eluting contact lens (MLF-CL).

#### **Table 1.** MLF-CL formulations. *2.3. Optical Coherence Topography*

**Formulation Name Drug: Polymer Ratio**  Commercial contact lens N/A MLF-CL2 2:25 MLF-CL4 4:25 MLF-CL8 8:25 To evaluate MLF-CL morphology and film thickness, all formulations were imaged using anterior segment optical coherence topography (AS-OCT, RTVue, Optovue, Fremont, CA, USA) to assess the morphology. Non-hydrated MLF-CLs were positioned with the convex side of the lens facing the OCT camera. Raster scanning imaging was used in four segments for each contact lens to obtain cross sectional images of the contact lens and drug-polymer film.

#### *2.3. Optical Coherence Topography 2.4. MLF-CL Release*

To evaluate MLF-CL morphology and film thickness, all formulations were imaged using anterior segment optical coherence topography (AS-OCT, RTVue, Optovue, Fremont, CA, USA) to assess the morphology. Non-hydrated MLF-CLs were positioned with the convex side of the lens facing the OCT camera. Raster scanning imaging was used in four segments for each contact lens to obtain cross sectional images of the contact lens and drug-polymer film. In vitro release of the MLF-CLs was conducted by hydrating MLF-CLs in 5 mL phosphate buffered saline (PBS) and placing in an incubator shaker at 37 ◦C at 64 rpm. At predetermined time points, the MLF-CLs were removed from PBS and immersed in fresh PBS. Release buffer aliquots were stored at 4 ◦C until drug concentration was quantified. Four lenses were used per formulation. Release quantification was performed by a colorimetric method.

#### *2.5. Water Content*

*2.5. Water Content*

*2.4. MLF-CL Release*  In vitro release of the MLF-CLs was conducted by hydrating MLF-CLs in 5 mL phosphate buffered saline (PBS) and placing in an incubator shaker at 37 °C at 64 rpm. At predetermined time points, the MLF-CLs were removed from PBS and immersed in fresh PBS. Release buffer aliquots were stored at 4 °C until drug concentration was quantified. Four lenses were used per formulation. Release quantification was performed by a colorimetric method. Water content of the MLF-CLs was performed by gravimetric method, according to ISO-18369-1 guidelines [30]. MLF-CLs were immersed in PBS until reaching equilibrium swelling, defined by a less than 5% change in mass in a 24 h period. Weight was recorded, then MLF-CLs were dried at 60 ◦C overnight and re-weighed. Weights were taken immediately after removal from the oven to prevent atmospheric moisture from influencing the results. All weights were recorded in duplicate. Four lenses were used per formulation. Percent water content was calculated by the following formula

$$\frac{\mathcal{W}\_{\rm s} - \mathcal{W}\_{\rm d}}{\mathcal{W}\_{\rm s}} \times 100\%$$

then MLF-CLs were dried at 60 °C overnight and re-weighed. Weights were taken immediately after removal from the oven to prevent atmospheric moisture from influencing the

ISO-18369-1 guidelines [30]. MLF-CLs were immersed in PBS until reaching equilibrium swelling, defined by a less than 5% change in mass in a 24 h period. Weight was recorded, where *Ws* = weight at equilibrium swelling and *Wd* = dry weight.

#### *2.6. Light Transmission*

Light transmission was measured through commercial contact lenses and the central aperture of the hydrated MLF-CL using SpectraMax M3 [31]. The average light transmission was calculated by averaging the transmission over the visible light spectrum (390–700 nm).

#### *2.7. Water Contact Angle*

MLF-CLs were hydrated in PBS until they reached equilibrium swelling. A drop of distilled water (about 5 µL) onto the surface via a syringe. Then, a high-resolution image was captured from the side using the Dino-Lite Edge camera. The contact angle for each formulation (*n* = 4 and 5 measurements per specimen) was determined using ImageJ software (NIH, Bethesda, MD, USA) as a function of time.

#### *2.8. Mechanical Properties*

The mechanical features of the MLF-CLs were determined at room temperature using a Mark-10 ESM 303 motorized test stand (Mark-10 Corporation, Copiague, NY, USA). Before analysis, samples were hydrated by immersing them in PBS for 2 days. The compression test was conducted on rings with 7 mm and 6 mm external and internal diameter, respectively and with a crosshead speed of 1 mm/min. The Compression Modulus was obtained from the linear derivative of the stress–strain curve in the low stiffness range (less than 10% strain). Three independent measurements were conducted for each group.

#### *2.9. Cytotoxicity*

L929 murine fibroblasts (American Tissue Culture Collection, Manassas, VA, USA) were used to assess the cytotoxicity of MLF-CLs. L929 cells were grown in Dulbecco's Modification of Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 4 mM L-glutamine. Cytotoxicity was measured by the minimum elution media method, described by ISO 10993-5 guidelines [32]. MLF-CLs or vehicle lenses (with the polymer film but no drug) were immersed in supplemented DMEM in a volume of 1.8 mL for each MLF-CL. The MLF-CLs were incubated at 37 ◦C for 24 h to capture eluted drugs and leachables. The elution media (extract) was applied neat to L929s plated in 96 well plates at 1 × 105 cells/mL the day prior. The cells with extract were incubated for 24 h. Cell viability was measured by CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) (Promega, Madison, WI, USA). Results were expressed as percentage viability of L929 controls that were not exposed to extract. Four lenses were used for each formulation, with six replicates per lens.

#### *2.10. Animals*

The study protocol #2021N000159 was approved on 7 July 2021 by the Institutional Animal Care and Utilization Committee at Schepens Eye Research Institute of Massachusetts Eye and Ear Infirmary (Boston, MA). All animals were treated according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research (ARVO Handbook, 1993). New Zealand white rabbits (NZW) (Charles River Laboratories, Boston, MA, USA) age four months, each weighing 3–5 kg, were used for the biocompatibility study. Only one eye in each NZW was used to prevent bilateral vision restriction. The contralateral eye was untreated and served as a control. NZWs were singly housed in a climate-controlled environment with free access to food and water during acclimation and throughout the experiment. NZWs were acclimated for seven days prior to experiments. In each of the studies, intramuscular injection of 30 mg/kg ketamine, 5 mg/kg xylazine, and 1 mg/kg acepromazine were used for anesthesia. 0.05 mg/kg Buprenorphine HCl was used for pain control prior to tarsorrhaphy.

Only MLF-CL4 was used for the biocompatibility study. Under anesthesia, NZWs had baseline photos taken by a Topcon DC-3 digital camera attachment for slit lamp. The MLF-CL was placed on the cornea, with care to place it under the nictating membrane. The MLF-CL was evaluated to ensure free movement on the cornea without fluting or bubbling.

To improve lens retention, eyes with contact lenses received a temporary tarsorrhaphy in which the eyelids were closed with one to two 5-0 Nylon sutures. NZWs were evaluated daily for signs of discomfort. After three days, the tarsorrhaphy and MLF-CL and reimaged. NZW eyes were evaluated for redness, edema, discharge, and fur matting. NZWs were euthanized with 120 mg/kg pentobarbital, and the corneas were removed from both eyes. The eyes were fixed in 10% formalin, embedded in paraffin, and cut into 10-micron sections in an anterior to posterior fashion so that the pupil and the optic nerve were in one section (PO section). Slides were stained with hematoxylin and eosin (H&E) to visualize ocular structures. sorrhaphy in which the eyelids were closed with one to two 5-0 Nylon sutures. NZWs were evaluated daily for signs of discomfort. After three days, the tarsorrhaphy and MLF-CL and re-imaged. NZW eyes were evaluated for redness, edema, discharge, and fur matting. NZWs were euthanized with 120 mg/kg pentobarbital, and the corneas were removed from both eyes. The eyes were fixed in 10% formalin, embedded in paraffin, and cut into 10-micron sections in an anterior to posterior fashion so that the pupil and the optic nerve were in one section (PO section). Slides were stained with hematoxylin and eosin (H&E) to visualize ocular structures.

NZWs were acclimated for seven days prior to experiments. In each of the studies, intramuscular injection of 30 mg/kg ketamine, 5 mg/kg xylazine, and 1 mg/kg acepromazine were used for anesthesia. 0.05 mg/kg Buprenorphine HCl was used for pain control prior

Only MLF-CL4 was used for the biocompatibility study. Under anesthesia, NZWs had baseline photos taken by a Topcon DC-3 digital camera attachment for slit lamp. The MLF-CL was placed on the cornea, with care to place it under the nictating membrane. The MLF-CL was evaluated to ensure free movement on the cornea without fluting or bubbling. To improve lens retention, eyes with contact lenses received a temporary tar-

#### *2.11. Statistics 2.11. Statistics*  For all continuous data, nonparametric descriptive statistics were used to determine

to tarsorrhaphy.

For all continuous data, nonparametric descriptive statistics were used to determine statistical significance. Kruskal–Wallis Test was calculated, and if significant, followed by pairwise comparisons to determine significant differences between groups. A *p*-Value of less than 0.05 was considered significant. statistical significance. Kruskal–Wallis Test was calculated, and if significant, followed by pairwise comparisons to determine significant differences between groups. A *p*-Value of less than 0.05 was considered significant.

#### **3. Results 3. Results**

#### *3.1. MLF-CL Was Successfully Encapsulated in Contact Lens Hydrogels 3.1. MLF-CL Was Successfully Encapsulated in Contact Lens Hydrogels*

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 5 of 15

Three dosages of MLF-CLs were created by changing the drug amount loaded in each lens while keeping PLGA concentration constant (Table 1). Drug-polymer (PLGA) films were created through solvent casting, then encapsulated in methalfilcon hydrogel using ultraviolet (UV) polymerization of hydroxyethylmethacrylate (HEMA) (Figure 1). When hydrated, the MLF-CLs expanded from 11.3 mm to 14.5 mm diameter and from 4 mm to 5 mm central aperture diameter and maintained a normal shape (Figure 1a). Anterior segment ocular coherence tomography (AS-OCT) images demonstrated uniform drug-polymer films completely within the methafilcon hydrogel and a clear aperture to preserve light transmission to the visual system (Figure 1b). Film thickness was roughly the same for all groups: MLF-CL2: 83.30 ± 6.08 µm, MLF-CL4: 81.11 ± 1.15 µm, MLF-CL8: 85.03 ± 5.62 µm) (*p* = 0.779, Kruskal–Wallis, *n* = 4). When imaged by OCT, no delamination of the film from the hydrogel was observed before hydration or after the lens was hydrated (Figure S1), indicating the film had sufficient adherence to the hydrogel that allow the film to maintain the same configuration during the hydration process which increased the diameter of the lens. Three dosages of MLF-CLs were created by changing the drug amount loaded in each lens while keeping PLGA concentration constant (Table 1). Drug-polymer (PLGA) films were created through solvent casting, then encapsulated in methalfilcon hydrogel using ultraviolet (UV) polymerization of hydroxyethylmethacrylate (HEMA) (Figure 1). When hydrated, the MLF-CLs expanded from 11.3 mm to 14.5 mm diameter and from 4 mm to 5 mm central aperture diameter and maintained a normal shape (Figure 1a). Anterior segment ocular coherence tomography (AS-OCT) images demonstrated uniform drug-polymer films completely within the methafilcon hydrogel and a clear aperture to preserve light transmission to the visual system (Figure 1b). Film thickness was roughly the same for all groups: MLF-CL2: 83.30 ± 6.08 µm, MLF-CL4: 81.11 ± 1.15 µm, MLF-CL8: 85.03 ± 5.62 µm) (*p* = 0.779, Kruskal–Wallis, *n* = 4). When imaged by OCT, no delamination of the film from the hydrogel was observed before hydration or after the lens was hydrated (Figure S1), indicating the film had sufficient adherence to the hydrogel that allow the film to maintain the same configuration during the hydration process which increased the diameter of the lens.

**Figure 1.** Miltefosine-eluting contact lens (MLF-CL). (**a**) Photographs of dry (left) and hydrated (right) contact lens. (**b**) An ocular coherence tomography (OCT) image of a section of a dry MLF-CL **Figure 1.** Miltefosine-eluting contact lens (MLF-CL). (**a**) Photographs of dry (left) and hydrated (right) contact lens. (**b**) An ocular coherence tomography (OCT) image of a section of a dry MLF-CL showing an encapsulated miltefosine-polymer film and central aperture to allow for unimpeded vision.

Drug release from MLF-CLs was studied in PBS at 37 ◦C. Because miltefosine aqueous solubility is relatively high (reported as 440 mg/mL [33]), frequent buffer changes were performed to preserve infinite sink conditions. All formulations demonstrated relatively constant state release through seven days (Figure 2). Cumulative 7-day miltefosine release ranged from 156.0 ± 4.7 µg (MLF-CL2) to 470.3 ± 39.8 µg (MLF-CL8) (Figure 2). We analyzed the miltefosine release kinetics from the MLF-CL using zero-order, first-order, Higuchi, and Korsmeyer-Peppas mathematical models (Table S1). Considering the highest

vision.

value of R2 -adjusted, the lowest Akaike Information Criterion (AIC) values and the largest Model Selection Criterion (MSC) [34], the release data fit well to the Korsmeyer-Peppas model for all formulations (Figure S2 and Table S1). The predicted release based on Korsmeyer-Peppas model was shown in Figure S2. These results indicated the diffusional release of miltefosine from contact lens, which was consistent with other drugs released from therapeutic contact lenses [31,35]. est value of R2-adjusted, the lowest Akaike Information Criterion (AIC) values and the largest Model Selection Criterion (MSC) [34], the release data fit well to the Korsmeyer-Peppas model for all formulations (Figure S2 and Table S1). The predicted release based on Korsmeyer-Peppas model was shown in Figure S2. These results indicated the diffusional release of miltefosine from contact lens, which was consistent with other drugs released from therapeutic contact lenses[31,35].

showing an encapsulated miltefosine-polymer film and central aperture to allow for unimpeded

Drug release from MLF-CLs was studied in PBS at 37 °C. Because miltefosine aqueous solubility is relatively high (reported as 440 mg/mL [33]), frequent buffer changes were performed to preserve infinite sink conditions. All formulations demonstrated relatively constant state release through seven days (Figure 2). Cumulative 7-day miltefosine release ranged from 156.0 ± 4.7 µg (MLF-CL2) to 470.3 ± 39.8 µg (MLF-CL8) (Figure 2). We analyzed the miltefosine release kinetics from the MLF-CL using zero-order, first-order, Higuchi, and Korsmeyer-Peppas mathematical models (Table S1). Considering the high-

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 6 of 15

**Figure 2.** In vitro miltefosine (MLF) release from MLF-CLs. (**A**) the cumulative release and (**B**) the daily release rate. Quantitative data are expressed as mean ± standard deviation (SD), *n* = 4/group per time point. **Figure 2.** In vitro miltefosine (MLF) release from MLF-CLs. (**A**) the cumulative release and (**B**) the daily release rate. Quantitative data are expressed as mean ± standard deviation (SD), *n* = 4/group per time point.

#### *3.2. MLF-CL Water Content Was Comparable to Commercial Contact Lenses 3.2. MLF-CL Water Content Was Comparable to Commercial Contact Lenses*

The water content of contact lenses is reported to have a significant impact on oxygen permeability and contact lens wearing comfort [36,37]. MLF-CL water content was assessed by the gravimetric method [38] and the results were compared to commercial methafilcon contact lenses (Kontur) (*n* = 4/group). Commercial contact lens had a water content (53 ± 1.6%) comparable to the reported value (55%) [39,40]. There was no significant (*p* = 0.49, Kruskal–Wallis Test) difference in the water content between MLF-CLs and commercial methafilcon lenses (Figure 3). The water content of contact lenses is reported to have a significant impact on oxygen permeability and contact lens wearing comfort [36,37]. MLF-CL water content was assessed by the gravimetric method [38] and the results were compared to commercial methafilcon contact lenses (Kontur) (*n* = 4/group). Commercial contact lens had a water content (53 ± 1.6%) comparable to the reported value (55%) [39,40]. There was no significant (*p* = 0.49, Kruskal–Wallis Test) difference in the water content between MLF-CLs and commercial methafilcon lenses (Figure 3). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 7 of 15

**Figure 3.** Equilibrium water content for miltefosine-eluting contact lenses (MLF-CLs) compared to commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples by Kruskal–Wallis Test. *p* = 0.49, *n* = 4. **Figure 3.** Equilibrium water content for miltefosine-eluting contact lenses (MLF-CLs) compared to commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples by Kruskal–Wallis Test. *p* = 0.49, *n* = 4.

The light transmittance properties of contact lenses are clearly important for visual performance. MLF-CL maintained light transmittance >95% through the central aperture

tween the commercial contact lenses and MLF-CL was negligible (*p* = 0.15, Kruskal–Wallis Test, *n* = 4 per group, Figure 4B), which was in agreement with our previous re-

**Figure 4.** (**A**) Light transmittance curve of commercial lens and central aperture of MLF-CL through the visible light spectrum (from 390 nm to 700 nm). (**B**) The average light transmission of MLF-CL, which was calculated by averaging the transmission over the visible light spectrum (390–700 nm). *n* = 4 lens per group. Quantitative data are expressed as mean ± standard deviation (SD). No significant difference in average light transmittance between the commercial contact lenses and MLF-CL

*3.4. Compression Modulus Was Similar between MLF-CLs and Commercial Contact Lenses*

Excessive stiffness in a contact lens can result in discomfort. Compression tests [42] were performed on the various MLF-CLs formulations to estimate their mechanical properties. The stress–strain curves of the different lenses were plotted, hence providing a visual display, indicating their strength and elasticity. MLF-CLs were compared to commercial CLs (*n* = 3/group, Figure 5). All groups were compared by Kruskal–Wallis Test.

*3.3. MLF-CL Light Transmittance Was comparable to Commercial Contact Lenses*

was observed (*p* = 0.15, Kruskal–Wallis Test).

ports[35,41].

#### *3.3. MLF-CL Light Transmittance Was comparable to Commercial Contact Lenses 3.3. MLF-CL Light Transmittance Was comparable to Commercial Contact Lenses*

significant statistical difference among samples by Kruskal–Wallis Test. *p* = 0.49, *n* = 4.

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 7 of 15

The light transmittance properties of contact lenses are clearly important for visual performance. MLF-CL maintained light transmittance >95% through the central aperture between 390 nm to 700 nm (Figure 4A). The difference in average light transmittance between the commercial contact lenses and MLF-CL was negligible (*p* = 0.15, Kruskal– Wallis Test, *n* = 4 per group, Figure 4B), which was in agreement with our previous reports [35,41]. The light transmittance properties of contact lenses are clearly important for visual performance. MLF-CL maintained light transmittance >95% through the central aperture between 390 nm to 700nm (Figure 4A). The difference in average light transmittance between the commercial contact lenses and MLF-CL was negligible (*p* = 0.15, Kruskal–Wallis Test, *n* = 4 per group, Figure 4B), which was in agreement with our previous reports[35,41].

**Figure 3.** Equilibrium water content for miltefosine-eluting contact lenses (MLF-CLs) compared to commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No

**Figure 4.** (**A**) Light transmittance curve of commercial lens and central aperture of MLF-CL through the visible light spectrum (from 390 nm to 700 nm). (**B**) The average light transmission of MLF-CL, which was calculated by averaging the transmission over the visible light spectrum (390–700 nm). *n* = 4 lens per group. Quantitative data are expressed as mean ± standard deviation (SD). No significant difference in average light transmittance between the commercial contact lenses and MLF-CL was observed (*p* = 0.15, Kruskal–Wallis Test). **Figure 4.** (**A**) Light transmittance curve of commercial lens and central aperture of MLF-CL through the visible light spectrum (from 390 nm to 700 nm). (**B**) The average light transmission of MLF-CL, which was calculated by averaging the transmission over the visible light spectrum (390–700 nm). *n* = 4 lens per group. Quantitative data are expressed as mean ± standard deviation (SD). No significant difference in average light transmittance between the commercial contact lenses and MLF-CL was observed (*p* = 0.15, Kruskal–Wallis Test).

#### *3.4. Compression Modulus Was Similar between MLF-CLs and Commercial Contact Lenses 3.4. Compression Modulus Was Similar between MLF-CLs and Commercial Contact Lenses*

Excessive stiffness in a contact lens can result in discomfort. Compression tests [42] were performed on the various MLF-CLs formulations to estimate their mechanical properties. The stress–strain curves of the different lenses were plotted, hence providing a visual display, indicating their strength and elasticity. MLF-CLs were compared to commercial CLs (*n* = 3/group, Figure 5). All groups were compared by Kruskal–Wallis Test. Excessive stiffness in a contact lens can result in discomfort. Compression tests [42] were performed on the various MLF-CLs formulations to estimate their mechanical properties. The stress–strain curves of the different lenses were plotted, hence providing a visual display, indicating their strength and elasticity. MLF-CLs were compared to commercial CLs (*n* = 3/group, Figure 5). All groups were compared by Kruskal–Wallis Test. Commercial CLs demonstrated the highest modulus at 491 ± 140 kPa. MLF-CL2 (220 ± 50 kPa), MLF-CL4 (296 ± 92 kPa) and MLF-CL8 (255 ± 30 kPa) with no significant differences among them (*p* = 0.1, Kruskal–Wallis Test). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 8 of 15 Commercial CLs demonstrated the highest modulus at 491 ± 140 kPa. MLF-CL2 (220 ± 50 kPa), MLF-CL4 (296 ± 92 kPa) and MLF-CL8 (255 ± 30 kPa) with no significant differences among them (*p* = 0.1, Kruskal–Wallis Test).

**Figure 5.** (**A**) Average stress–strain curves and (**B**) Compression modulus for miltefosine-eluting contact lenses (MLF-CLs) compared to commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples. by Kruskal– Wallis Test. *p* = 0.1, *n* = 3. **Figure 5.** (**A**) Average stress–strain curves and (**B**) Compression modulus for miltefosine-eluting contact lenses (MLF-CLs) compared to commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples. by Kruskal– Wallis Test. *p* = 0.1, *n* = 3.

Water contact angle is a measure of the wettability of a surface. Poor wettability can

used method for biomaterials [44]. All contact lenses were cut to lie flat to ensure the most accurate measurements. Compared to commercial contact lenses (26.2 ± 4.0°), there were no significant differences (*p* = 0.06, Kruskal–Wallis Test) among the MLF-CL groups (*n* =

**Figure 6.** Water contact angle of miltefosine-eluting contact lenses (MLF-CLs) and commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant sta-

tistical difference among samples by Kruskal–Wallis Test. *p* = 0.06, *n* = 4.

4/group) (Figure 6).

*3.5. MLF-CL Surface Wettability was Comparable to Commercial Contact Lenses*

#### *3.5. MLF-CL Surface Wettability was Comparable to Commercial Contact Lenses 3.5. MLF-CL Surface Wettability was Comparable to Commercial Contact Lenses*

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 8 of 15

among them (*p* = 0.1, Kruskal–Wallis Test).

MLF-CL2 MLF-CL4 MLF-CL8

Wallis Test. *p* = 0.1, *n* = 3.

Water contact angle is a measure of the wettability of a surface. Poor wettability can break up the tear film, leading to dry eyes [36,43]. Water contact angle was measured in MLF-CLs and commercial contact lenses using sessile drop technique, the most commonly used method for biomaterials [44]. All contact lenses were cut to lie flat to ensure the most accurate measurements. Compared to commercial contact lenses (26.2 ± 4.0◦ ), there were no significant differences (*p* = 0.06, Kruskal–Wallis Test) among the MLF-CL groups (*n* = 4/group) (Figure 6). Water contact angle is a measure of the wettability of a surface. Poor wettability can break up the tear film, leading to dry eyes [36,43]. Water contact angle was measured in MLF-CLs and commercial contact lenses using sessile drop technique, the most commonly used method for biomaterials [44]. All contact lenses were cut to lie flat to ensure the most accurate measurements. Compared to commercial contact lenses (26.2 ± 4.0°), there were no significant differences (*p* = 0.06, Kruskal–Wallis Test) among the MLF-CL groups (*n* = 4/group) (Figure 6).

**Figure 5.** (**A**) Average stress–strain curves and (**B**) Compression modulus for miltefosine-eluting contact lenses (MLF-CLs) compared to commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples. by Kruskal–

Commercial CLs demonstrated the highest modulus at 491 ± 140 kPa. MLF-CL2 (220 ± 50 kPa), MLF-CL4 (296 ± 92 kPa) and MLF-CL8 (255 ± 30 kPa) with no significant differences

**Figure 6.** Water contact angle of miltefosine-eluting contact lenses (MLF-CLs) and commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples by Kruskal–Wallis Test. *p* = 0.06, *n* = 4. **Figure 6.** Water contact angle of miltefosine-eluting contact lenses (MLF-CLs) and commercial contact lenses. Quantitative data are expressed as mean ± standard deviation (SD). No significant statistical difference among samples by Kruskal–Wallis Test. *p* = 0.06, *n* = 4.

#### *3.6. MLF-CL Exhibited Dose-Dependent In Vitro Cytotoxicity to L929 Murine Fibroblasts*

Because miltefosine or MLF-CL leachables such as unreacted monomer, photoinitiator, or residual solvent could potentially cause toxicity, we tested the cytotoxicity of MLF-CLs by the minimum elution media method, where the extract from hydrated MLF-TCLs was exposed to L929 murine fibroblasts [45,46]. The L929 murine fibroblast cells are highly proliferative and frequently used in cytotoxicity testing to evaluate cellular viability and proliferation [47]. Results were compared to L929 cells not exposed to extract. Vehicle lenses showed no cytotoxicity (98.7 ± 7.3%). MLF-CLs showed viability that was inversely proportional to MLF content, with viabilities of 90.6 ± 3.3%, 72.7 ± 4.8%, and 29.8 ± 1.9% for MLF-CL2, MLF-CL4, and MLF-CL8, respectively (Figure 7).

#### *3.7. MLF-CLs Demonstrated Suitable Biocompatibility In Vivo*

Potential risks with contact lens wear include corneal edema, cornea abrasions, and microbial keratitis and there is also the risk of local or systemic toxicity from topically delivered miltefosine. Normal New Zealand White (NZW) rabbits were used to assess biocompatibility as rabbits are the lowest order mammal that can wear a contact lens. In addition, NZW eye dimensions are comparable to those of a human [48]. MLF-CL4s were chosen to be evaluated in vivo as they had demonstrated suitable physicochemical properties similar to other commercial contact lenses while maintaining an acceptable cytotoxicity level. NZWs (*n* = 3, two females, one male) wore the MLF-CL4s continuously for three days. One eye showed erythema throughout the conjunctiva, on day 3. In the other two rabbits, no abnormalities were seen in daily observations during the lens wear period. No erythema, discharge, lid edema, or epithelial defects were noted on slit lamp exam after three days of continuous wear.

Pathology revealed normal corneas and irises in all NZWs. No changes in the corneal epithelium, stromal lamellae, Descemet's membrane, and endothelium were observed (Figure 8). Results were compared to the contralateral eyes of each rabbit, showing no difference between eyes that wore the MLF-CL and untreated eyes. proliferation [47]. Results were compared to L929 cells not exposed to extract. Vehicle lenses showed no cytotoxicity (98.7 ± 7.3%). MLF-CLs showed viability that was inversely proportional to MLF content, with viabilities of 90.6 ± 3.3%, 72.7 ± 4.8%, and 29.8 ± 1.9% for MLF-CL2, MLF-CL4, andMLF-CL8, respectively (Figure 7).

Because miltefosine or MLF-CL leachables such as unreacted monomer, photoinitiator, or residual solvent could potentially cause toxicity, we tested the cytotoxicity of MLF-CLs by the minimum elution media method, where the extract from hydrated MLF-TCLs was exposed to L929 murine fibroblasts [45,46]. The L929 murine fibroblast cells are highly proliferative and frequently used in cytotoxicity testing to evaluate cellular viability and

*3.6. MLF-CL Exhibited Dose-Dependent In Vitro Cytotoxicity to L929 Murine Fibroblasts*

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 9 of 15

**Figure 7.** Cell viability of L929s with eluted media from miltefosine-eluting contact lenses (MLF-CLs). Results are expressed as percent viability of untreated L929s. Cell viability for MLF-CLs compared to vehicle lenses. \* *p* = 0.017, \*\* *p* < 0.001 by Kruskal–Wallis Test with Bonferroni correction. *n*  **Figure 7.** Cell viability of L929s with eluted media from miltefosine-eluting contact lenses (MLF-CLs). Results are expressed as percent viability of untreated L929s. Cell viability for MLF-CLs compared to vehicle lenses. \* *p* = 0.017, \*\* *p* < 0.001 by Kruskal–Wallis Test with Bonferroni correction. *n* = 4. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 10 of 15

rabbits, no abnormalities were seen in daily observations during the lens wear period. No erythema, discharge, lid edema, or epithelial defects were noted on slit lamp exam after three days of continuous wear. **Figure 8.** Hematoxylin and Eosin(H&E) stained corneas for New Zealand white rabbits (NZW) eyes wearing miltefosine-eluting contact lenses (MLF-CL4) compared to the contralateral eye (untreated). **Figure 8.** Hematoxylin and Eosin(H&E) stained corneas for New Zealand white rabbits (NZW) eyes wearing miltefosine-eluting contact lenses (MLF-CL4 ) compared to the contralateral eye (untreated).

#### Pathology revealed normal corneas and irises in all NZWs. No changes in the corneal **4. Discussion 4. Discussion**

epithelium, stromal lamellae, Descemet's membrane, and endothelium were observed (Figure 8). Results were compared to the contralateral eyes of each rabbit, showing no difference between eyes that wore the MLF-CL and untreated eyes. In this work, we have encapsulated miltefosine in a contact lens hydrogel. This device provides sustained release of miltefosine for one week and may be of use in the treatment of AK. To our knowledge, this is the first report of a miltefosine sustained release system for the treatment of ocular disease. In this work, we have encapsulated miltefosine in a contact lens hydrogel. This device provides sustained release of miltefosine for one week and may be of use in the treatment of AK. To our knowledge, this is the first report of a miltefosine sustained release system for the treatment of ocular disease.

PLGA was chosen due to its safety and excellent sustained drug release profiles [49]. PLGA is a component of numerous FDA- and European Medicine Agency (EMA)- approved drug delivery devices. The ratio of lactide to glycolide of 85:15 in PLGA was chosen because higher lactide content is associated with a decrease in degradation rate and drug release rate [49]. PLGA can degrade into its two monomeric constituents (lactic acid and glycolic acid) via hydrolysis reactions, which in turn can be easily metabolized via the Krebs cycle. Therefore, PLGA produces minimal systemic toxicity when used in biological applications [50], and showed no toxicity in our studies. The films were made by PLGA was chosen due to its safety and excellent sustained drug release profiles [49]. PLGA is a component of numerous FDA- and European Medicine Agency (EMA)- approved drug delivery devices. The ratio of lactide to glycolide of 85:15 in PLGA was chosen because higher lactide content is associated with a decrease in degradation rate and drug release rate [49]. PLGA can degrade into its two monomeric constituents (lactic acid and glycolic acid) via hydrolysis reactions, which in turn can be easily metabolized via the Krebs cycle. Therefore, PLGA produces minimal systemic toxicity when used in biological applications [50], and showed no toxicity in our studies. The films were made by solvent

solvent casting and lathing, but other approaches could be employed, such as 3D printing

release of miltefosine for 24 h [53]. In contrast, our MLF-CLs achieved a stable release rate over a period of 7 consecutive days. The sustained release of MLF-CLs may be attributed to the high partition coefficient (logP of 3.8) of miltefosine and interaction between drug and PLGA [54]. Miltefosine was miscible with PLGA in the drug-polymer film (Figure 1), indicating the interaction between drug and PLGA. Considering the hydrophobic properties of miltefosine and PLGA, this suggests drug-polymer interactions were due to this shared hydrophobicity with charge interactions as a secondary effect. The drug-polymer interaction can be investigated by Fourier-transform infrared spectroscopy (FTIR) and Differential Scanning Calorimeter (DSC) [55,56]. These results were consistent with our previous reports that PLGA films in contact lenses were able to sustain the release of hydrophobic drugs such as dexamethasone and latanoprost [35,41,57,58]. While AK often requires a treatment duration of weeks to months, extended wear of a single contact lens is limited to seven days to prevent complications from contact lens overwear, including corneal edema and microbial keratitis. In practice, a patient with AK could wear consecutive MLF-CLs—exchanged once weekly by an ophthalmologist—until AK is eradicated. Previous studies have reported a minimum amebicidal concentration (MAC) of 16 µg/mL for clinical and environmental isolates [59–61]. The formulations MLF-CL2, MLF-CL4 and MLF-CL8 released 24, 85 and 151 µg of miltefosine on the first day, respectively. The daily release rate thereafter ranged from 17-26 µg/day for MLF-CL2, 24-42 µg/day for MLF-CL4, and ranged from 41-89 µg/day for MLF-CL8 (Figure 2B) after one week of testing, well above the MAC. Based on the design of contact lens drug delivery, the fluid exchange in the post-lens tear film that exists between the cornea and the contact lens has

In a miltefosine-loaded nanostructured lipid carriers release system [52], most drugs

of the film and producing contact lenses from molds [51].

casting and lathing, but other approaches could be employed, such as 3D printing of the film and producing contact lenses from molds [51].

In a miltefosine-loaded nanostructured lipid carriers release system [52], most drugs were released within a few hours. Miltefosine-loaded alginate nanoparticles sustained the release of miltefosine for 24 h [53]. In contrast, our MLF-CLs achieved a stable release rate over a period of 7 consecutive days. The sustained release of MLF-CLs may be attributed to the high partition coefficient (logP of 3.8) of miltefosine and interaction between drug and PLGA [54]. Miltefosine was miscible with PLGA in the drug-polymer film (Figure 1), indicating the interaction between drug and PLGA. Considering the hydrophobic properties of miltefosine and PLGA, this suggests drug-polymer interactions were due to this shared hydrophobicity with charge interactions as a secondary effect. The drug-polymer interaction can be investigated by Fourier-transform infrared spectroscopy (FTIR) and Differential Scanning Calorimeter (DSC) [55,56]. These results were consistent with our previous reports that PLGA films in contact lenses were able to sustain the release of hydrophobic drugs such as dexamethasone and latanoprost [35,41,57,58]. While AK often requires a treatment duration of weeks to months, extended wear of a single contact lens is limited to seven days to prevent complications from contact lens overwear, including corneal edema and microbial keratitis. In practice, a patient with AK could wear consecutive MLF-CLs—exchanged once weekly by an ophthalmologist—until AK is eradicated.

Previous studies have reported a minimum amebicidal concentration (MAC) of 16 µg/mL for clinical and environmental isolates [59–61]. The formulations MLF-CL2, MLF-CL4 and MLF-CL8 released 24, 85 and 151 µg of miltefosine on the first day, respectively. The daily release rate thereafter ranged from 17-26 µg/day for MLF-CL2, 24–42 µg/day for MLF-CL4, and ranged from 41-89 µg/day for MLF-CL8 (Figure 2B) after one week of testing, well above the MAC. Based on the design of contact lens drug delivery, the fluid exchange in the post-lens tear film that exists between the cornea and the contact lens has been shown to be slower than fluid exchange on the surface of the eye [62,63], which may improve the drug bioavailability compared to topical eye drops. In our previous study with a latanoprost contact lens, we found that contact lenses with drug-polymer films had high correlation coefficients between drug release in vitro and drug absorption in vivo [57]. We expect that the amount of miltefosine released by MLF-CLs in the eye may reach therapeutic amounts in vivo.

The light transmittance of central aperture was above 95% for all three formulation and was comparable to commercial lens, though the film-embedded part of the MLF-CL showed semitransparency (Figure 1). We also have the flexibility to increase the MLF-CL central aperture size to further increase visual performance.

Wettability and mechanical properties of the lenses are critical for the physiological compatibility, handling, durability, and comfort of wearing the lenses. In fact, oxygen diffusivity and water content are key parameters influencing the comfort and safety of a lens [36]. MLF-CLs showed similar water content compared to commercial methafilcon contact lenses of about 53% water uptake. At a range of 26–33◦ , MLF-CL wettability was also similar to commercial contact lenses. One of the limitations to our study is that we did not directly measure oxygen transmittance, which has been associated with wearing comfort.

The modulus range for the MLF-CLs we measured was between 210 kPa and 300 kPa. We found that MLF-CLs are softer than commercial contact lenses (490 kPa) as they showed a significantly lower modulus. This decrease in modulus is most likely the result of increased chain mobility in the contact lens hydrogel. Nonetheless, the modulus of MLF-CL is still within the acceptable range of values reported in the literature for the currently available contact lens materials (300 to 600 kPa) [64].

MLF-CL showed cell viability inversely proportional to dose: MLF-CL2 had the highest cell viability (91%) and MLF-CL8 had the lowest cell viability (36%). Given that the vehicle lens had cell viability of close to 100%, the decreased cell viability is solely associated with increased miltefosine dose. Nonetheless, MLF-CL4 had an acceptable level (73%) of cell survival by ISO standards [65]. In addition, Miltefosine was significantly less toxic than 0.02% (200 µg/mL) chlorhexidine, a standard concentration of chlorhexidine drops used to treat AK [66]. In our own studies, chlorhexidine showed significant toxicity at 0.005% (50 µg/mL), compared to miltefosine alone, which did not demonstrate cytotoxicity until 90 µg/mL (Figure S3). This is higher than the believed efficacious dose of 65.12 µg/mL. Previous in vitro studies on anti-AK drugs have shown low cell viability of 21% with 0.02% chlorhexidine (CHX) and 38% with both 0.02% polyhexamethylene biguanide (PHMB) and 0.01% (100 µg/mL) propamidine isethionate (PI). Given the favorable physicochemical properties of MLF-CL4, combined with minor in vivo toxicity, additional formulations can be investigated with changing miltefosine concentrations to reduce cytotoxicity. We also have the flexibility to adjust the MLF-CL central aperture size to achieve this purpose.

In the biocompatibility experiment for this study, one of three rabbits exhibited adverse effects after three days of wear. Miltefosine acts as an immunomodulator, activating Type 1 T-helper cells (Th1) which in turn increase the recipient's inflammatory response. Cytokines, particularly interferon gamma and interleukin 12 have been observed to be elevated with oral miltefosine use [67]. However, pathology did not reveal any abnormalities in the cornea and iris, indicating the adverse effects of miltefosine were self-limiting. In clinical practice, the severe inflammation reaction is alleviated by prescribing oral and/or topical steroids [24], though the practice is often contested, as steroids can dampen the body's natural immune response [68,69]. Topical steroids could be prescribed, or potentially include steroids in the MLF-CL. In vivo and in vitro correlation can be poor, which also supports testing in vivo biocompatibility. More research is needed (i.e., evaluating longterm outcomes of MLF-CL wear) to determine the appropriate course of action.

#### **5. Conclusions**

We incorporated the miltefosine-PLGA film into a commercially available hydrogel material to form a delivery system that can slowly release miltefosine. MLF-CL's physical properties were not significantly different from commercial contact lenses in terms of light transmittance, water content, and wettability. This ocular drug delivery system provides a slow and steady release of miltefosine over the course of a week and has the safety profile to serve as a promising drug treatment platform for AK. More critical efforts are needed to develop an efficient and safe MLF-CL device that can be validated in animals and clinics. In accordance with the outcomes of this investigation, we envision that the MLF-CL4 lens is very suitable for further in vivo and clinical studies, as it shows comparable physicomechanical properties to the commercial contact lens, optimal biocompatibility, and an enhanced and sustained drug release over a period on one week.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14122750/s1, Figure S1: The OCT image of hydrated MLF-CL; Figure S2: In vitro release profiles (actual cumulative release %) of MLF-CL and mathematical modeling (predicted release %) of the miltefosine release profiles. The in vitro release profiles of miltefosine from MLF-CL fit to the Korsmeyer-Peppas model. Data are shown as the means ± SD, n = 4 for each group; Figure S3: Cell viability of L929s subjected to different concentrations of (A) Miltefosine (µg/mL) and (B) Chlorhexidine free base drug (µg/mL); Table S1: Mathematical model examination for the miltefosine release from MLF-CL. R2 -adjusted, Akaike Information Criterion (AIC) and the Model Selection Criterion (MSC) employed for model selection (n = 4). The best model possesses the highest value of R2-adjusted, the lowest AIC value and the largest MSC [34].

**Author Contributions:** Conceptualization: L.C., L.K., A.E.R. and J.B.C. Investigation: L.C., L.K., A.E.R., W.F., N.B., S.S., L.N.K., J.B.C. Analysis: L.C., L.K., A.E.R., W.F., N.B. and J.B.C. Writing— Original draft preparation: L.C., L.K., A.E.R., W.F. Visualization: L.K., W.F., A.E.R., L.C., L.L. Writing review and editing: L.C., L.K., A.E.R., W.F., N.B., L.N.K., D.S.K., J.B.C. Supervision: J.B.C. Project administration: J.B.C. Funding acquisition: L.C. and J.B.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** L.C. was supported by the China Scholarship Council and the National Nature Science Foundation of China (No. 81960183). This project was funded by NIH 1R01 EY026640 (JBC) and P30EY003790 (Schepens Eye Research Institute Core).

**Institutional Review Board Statement:** The animal protocol was approved by the Schepens Eye Research Institute Institutional Animal Care and Use Committee (Protocol 2021N000159, approved on 7 July 2021.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Miele MacMillan and Honney Thomas for assistance with animal studies, Menglu Yang for cell culture assistance, Mengqiu Huang for assistance with schematic illustration, and Jerry Niederkorn for experimental suggestions.

**Conflicts of Interest:** J.B.C. and D.S.K. have a financial interest in Theroptix, a company developing a contact lens drug delivery system. They are inventors of the technology, co-founders, and serve as consultants. J.B.C.'s interests were reviewed and are managed by Massachusetts Eye and Ear and Mass General Brigham Healthcare in accordance with their conflict of interest policies. D.S.K.'s interests are managed by Boston Children's Hospital in accordance with their conflict of interest policies.

### **References**

## *Article* **Besifloxacin Nanocrystal: Towards an Innovative Ophthalmic Preparation**

**José Izo Santana da Silva de Jesus 1,† , Felipe Rebello Lourenço 1 , Kelly Ishida 2 , Thayná Lopes Barreto 2 , Valdir Carlos Avino 3 , Edson dos Santos Neto 4 and Nádia Araci Bou-Chacra 1,\* ,†**

**Abstract:** Bacterial conjunctivitis significantly impacts public health, including more than one-third of eye diseases reported worldwide. It is an infection caused by various aerobic and anaerobic bacteria and is highly contagious. Therefore, it has a high incidence of bacterial resistance to the antibiotics commonly used for treatment. Among the most recent antibiotics, besifloxacin is a fourth-generation fluoroquinolone antibiotic indicated exclusively for topical ophthalmic use. Due to its importance in treating bacterial conjunctivitis and its low solubility in water, limiting its efficacy, a nanotechnologybased drug delivery preparation was developed to overcome this hurdle. Besifloxacin nanocrystals were prepared by small-scale wet milling and response surface methodology, using Povacoat® as a stabilizer. The particle's average hydrodynamic diameter (Z-ave) was approximately 550 nm (17 times smaller than raw material), with a polydispersity index (PdI) of less than 0.2. The saturation solubility increased about two times compared to the raw material, making it possible to increase the dissolution rate of this drug substance, potentially improving its bioavailability and safety. The optimized preparation was stable under an accelerated stability study (90 days). The Z-ave, PZ, PdI, and content did not alter significantly during this period. Furthermore, the 0.6% m/m besifloxacin nanocrystals at the maximum dose and the Povacoat® stabilizer did not show toxicity in *Galleria mellonella* larvae. The innovative ophthalmic preparation minimum inhibitory concentration (MIC) was 0.0960 µg/mL and 1.60 µg/mL against *Staphylococcus aureus* and *Pseudomonas aeruginosa*, respectively, confirming in vitro efficacy. Therefore, besifloxacin nanocrystals revealed the potential for reduced dosing of the drug substance, with a minor occurrence of adverse effects and greater patient adherence to treatment.

**Keywords:** nanocrystals; conjunctivitis; besifloxacin; Povacoat®; fluoroquinolones

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

### **1. Introduction**

Conjunctivitis refers to the inflammatory process of the conjunctiva and affects all age groups [1]. Bacterial conjunctivitis is responsible for more than one-third of eye complications worldwide, causing pain and discomfort to patients, decreasing their quality of life, and increasing health care costs [2–5]. Alfonso and colleagues [6] report that the costs of treating conjunctivitis ranged from 377 to 875 million dollars annually in the United States.

Bacterial conjunctivitis is highly contagious and can be caused by aerobic and anaerobic bacteria. It also includes other common species such as *Neisseria gonorrhoeae*, *Neisseria*

**Citation:** Jesus, J.I.S.d.S.d.; Lourenço, F.R.; Ishida, K.; Barreto, T.L.; Avino, V.C.; Neto, E.d.S.; Bou-Chacra, N.A. Besifloxacin Nanocrystal: Towards an Innovative Ophthalmic Preparation. *Pharmaceutics* **2022**, *14*, 2221. https:// doi.org/10.3390/pharmaceutics 14102221

Academic Editors: Francisco Javier Otero-Espinar and Anxo Fernández Ferreiro

Received: 10 September 2022 Accepted: 5 October 2022 Published: 18 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

15

*meningitides*, *Pseudomonas*, *Proteus*, and *Corynebacterium* [1,2,4]. Bacterial infections, if not treated properly, can have potentially serious consequences, including irreversible vision complications such as permanent loss [7]. Furthermore, in some cases, such as after glaucoma surgery, the contagion of bacterial conjunctivitis increases the patient's risk of developing endophthalmitis [8]. This devastating complication can cause permanent vision loss [9,10].

In the case of more severe complications, such as the development of mucopurulent exudate, treatment with antibiotics should be immediate, including several classes of antibiotics, such as aminoglycosides, combinations of polymyxin B, macrolides, sulfonamides, and the fluoroquinolones [2,4]. The indication of the fluoroquinolone class as topical ophthalmic was only from 1990 onwards and brought significant therapeutic advantages, as it has a broad spectrum and low toxicity. However, many of these therapeutic classes are no longer effective or show bacterial resistance [11]. In this context, besifloxacin, a recent member of fourth-generation fluoroquinolones, has significant advantages. This antimicrobial was developed exclusively for topical ophthalmic use. Unlike other fluoroquinolones, besifloxacin is not used for treating other infections, and this drug substance is the only one in its class to present a lower risk of developing resistance [2,5].

The synthesis of besifloxacin was initiated by a Japanese company, and in 2009 it was approved by the Food and Drug Administration (FDA) for the treatment of bacterial conjunctivitis in the United States; two years later, it was approved by ANVISA in Brazil [12,13]. Besifloxacin shows low water solubility (0.143 mg/mL) and is marketed as besifloxacin hydrochloride by Bausch & Lomb Incorporated under the trade name Besivance®, 0.6% ophthalmic suspension [5,14,15].

Conventional ophthalmic preparations (drops) are rapidly removed from the ocular surface due to eye protection mechanisms. Therefore, the retention time of drugs in the eye is minimal with consequently low bioavailability, generally less than 5% [16,17]. A different nano-based besifloxacin delivery was developed to overcome these challenges [17–20]. However, there are no besifloxacin nanocrystals reported in the literature. Nanocrystal increases the surface area to volume ratio of the drug substance, enhancing its saturation solubility, dissolution velocity, and adhesiveness when compared to the drug on the micrometric scale, with a consequent increase in bioavailability [21–25]. Given these benefits, the global nanotechnology market for drug delivery is projected to reach US\$ 164.1 billion by 2027 [26], showing that the global nanotechnology market for drug delivery is projected to reach US\$ 164.1 billion by 2027. Nanocrystals alone should reach US\$ 83.1 billion in this period, with a growth of 21.9% for the next years.

In general, drug nanocrystals are prepared in dispersion media with the addition of stabilizers (surfactants and polymers), resulting in a colloidal state referred to as nanosuspensions [21,22]. The choice of stabilizer is complex and critical in the development stage of nanocrystals. Design space, a strategic tool in the QbD concept, allows for understanding and establishing multivariate combinations and interactions of input variables for the proper development of the product or process [27,28].

Therefore, this study aims to develop besifloxacin nanocrystals using a spatial design approach with subsequent physicochemical characterization, in vivo toxicity (*Galleria mellonella model*), and in vitro efficacy by determining the minimum inhibitory concentration against two main bacteria causing conjunctivitis.

#### **2. Materials and Methods**

#### *2.1. Materials*

Besifloxacin (99.93%, MW 430.301 g/mol, Log P 0.54, water solubility: 0.143 mg/mL) was purchased from Jinan Shengqi Pharmaceutical Co., Ltd. (Jinan China). Povacoat® (POVA) was donated from DAIDO Takeshima Nishiyodogawa-Ku, Osaka, Japan). Polysorbate 80, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzyldimethylhexadecylammonium chloride 16-BAC), and chitosan (CS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poloxamer 188 (P188), Poloxamer 407 (P407), Poloxamer HS 15 (HS

15), tocopherol polyethylene glycol succinate (TPGS), and Kollicoat® IR (KLT) were purchased from BASF (Ludwigshafen am Rhein, Germany). Triethylamine, phosphoric acid, acetonitrile, and phosphate buffer solution pH 7.0 (Certipur®) were purchased from Merck (Darmstadt, Germany). The product Besivance® (0.6% ophthalmic suspension) (Bausch & Lomb Incorporated, Bridgewater, NJ, USA) was obtained from a local market. Milli-Q® Integral 10 ultrapure water (Merck KGaA, Darmstadt, Germany) was used for analytical purposes.

#### *2.2. Drug Analysis*

#### 2.2.1. Particle Size Determination by Laser Diffraction

The particle size of besifloxacin (raw material) was determined and compared with the commercial product (Besivance®), for which the Beckman Coulter™ LS 13 320 laser diffraction particle size analyzer equipped with a ULM module (Universal Liquid Module) was used (Beckman Coulter Inc., Brea, CA, USA). A suspension of the drug was prepared in phosphate buffer pH 7.0, and the commercial product (Besivance®) was used directly for analysis. Results represent the average of three readings.

#### 2.2.2. Determination of Minimum Inhibitory Concentration (MIC)

The MIC of besifloxacin (raw material) against *Staphylococcus aureus* ATCC® 23235™ and *Pseudomonas aeruginosa* ATCC® 9027™ were determined by the microdilution method. Posteriorly, tubes containing the serial dilutions (0.125 to 60 µg/mL) were incubated in an oven at 37 ◦C for 24 h. The determination of the inhibition and/or growth of microorganisms was performed by spectrophotometer [29].

### *2.3. Preparation of Besifloxacin Nanocrystals Using Small-Scale Wet Bead Milling*

#### 2.3.1. Exploratory Tests for Stabilizer and Statistical Variables Selection

The besifloxacin nanocrystals were obtained using a small-scale top-down approach by wet bead milling, according to Romero, Keck, and Müller [30]. The system was composed of three magnetic stirring bars (5 mm each) arranged on top of each other in a 10 mL glass vial, stirred by a magnetic stirring plate (IKA®-WERKE, Staufen im Breisgau, Germany). In the first exploratory assay, the proposed formulation consisted of 0.6 wt% of besifloxacin (BSF) and 0.6 wt% of conventional stabilizers (1:1 wt ratio). In the second part, the same drug concentration was maintained (0.6 wt%); however, new stabilizers and different proportions were tested. All formulations had their total weight adjusted to 10 g with buffer solution pH 7.0 (dipotassium hydrogen phosphate/disodium hydrogen phosphate). Additionally, 30% of the vial volume was filled with 3.0 g (*w*/*w*) zirconium beads (size of 0.1 mm) (Retsch, Germany).

Initially, each tested stabilizer was weighed and transferred to the vial containing half of the planned buffer solution, and the system was stirred for 10 min to ensure total solubilization. In sequence, the besifloxacin was weighed and added to the formulation and the remaining buffer solution to complete 10 g of nanosuspension. The milling process was at 1200 rpm for 4 days for all stabilizers. The measurements of average hydrodynamic diameter (Z-ave) and polydispersity index (PdI) were determined within 24 h of the milling process.

The formulations whose stabilizers allowed particle size reduction to nanometer-scale were kept at room temperature. Z-ave and PdI measurements were determined after 7 and 15 days to verify the maintenance of particle size stability. Additionally, as a part of the third preliminary study, the processing time (24 and 96 h) and the number of magnetic bars (2 and 3) were tested with the stabilizer Povacoat®, chosen for the optimization step.

#### 2.3.2. Nanosuspension Optimization Process Applying Box–Behnken Experiment Design

The Box–Behnken design refers to a response surface design without considering the built-in full factorial or fractional design (Minitab® 18). For this model, at least three factors were considered. In this sense, the exploratory studies allowed to establish the agitation speed as a critical parameter of the process, as well as the stabilizer (Povacoat®), the drug (besifloxacin), and their concentrations, to optimize the formulation. These independent variables were selected for the experiment, as follows in Table 1. Thus, this experiment was used to evaluate the responses of the dependent variable, average hydrodynamic diameter (Z-ave).

**Table 1.** Independent variables and levels selected for Box–Behnken design.

#### 2.3.3. Mathematical Model Verification

The mathematical equation derived from the response surface analysis determined the besifloxacin nanosuspensions formulations for the statistical model verification. These formulations were prepared using the conditions described in this section on the nanosuspension optimization process applying a Box–Behnken experiment design. The Z-ave measurement was determined at the end of this period.

#### *2.4. Dynamic Light Scattering and Zeta Potential*

The nanocrystals Z-ave and polydispersity indexes (PdI) were determined by photon correlation spectroscopy using Zetasizer Nano SZ90 (Malvern Instruments, Malvern, UK). Light scattering was monitored at 25 ◦C at an angle of 90◦ . The samples were previously diluted in the besifloxacin saturated solution (ratio of 1:24) for a total of 1 mL, considering an instrument sensitivity range. The measurements used disposable polystyrene cells. The analysis was performed once for each nanosuspension, and the software performed three runs.

The ZP was carried out by electrophoretic light scattering using the same equipment. Three measurements were taken for each sample, each comprising 14 accumulations. The measurements were taken at 25 ◦C before dilution with buffer solution pH 7.0 (conductivity = 2238 µS/cm). The applied field force was 20 V/cm, and the results were obtained from a Henry equation [31].

#### *2.5. Distribution and Particle Size by Laser Diffraction (LD)*

The distribution and particle size of the besifloxacin nanosuspension (0.6 w%) were performed by low-angle laser scattering using the Mastersizer 2000 equipment (Malvern Instruments, Malvern, UK), equipped with a Hydro 2000MU module (Malvern Instruments), Malvern, UK). The equipment was programmed for the wet method with a measurement range from 50 nm to 1 mm, using pH 7.0 phosphate buffer for dispersion. Granule refractive index: 1.67. The results were presented based on the volume distribution (D10, D50 and D90), which indicates the percentage of existing particles below the given size (nm).

#### *2.6. Density, Viscosity, and pH*

The density was determined using a DMA 4500 M densimeter (Anton Paar GmbH, Graz, Austria). For the measurement, the sample was placed directly in the equipment, in enough quantity to fill the hose/channel. The density was determined concerning water at 25 ◦C.

The viscosity was performed using an LVDV-I PRIME viscometer (Brookfield, Middleboro, MA, USA), equipped with an LV-3 spindle (63), and the speed was 50 rpm. For testing, 15 mL of the nanosuspension was transferred to the SC4-13RPY sample container (Brookfield, Middleboro, MA, USA).

The pH was determined with an Orion™ Versa Star Pro™ potentiometer (Thermo Fisher Scientific, Waltham, MA, USA). All these analyses were performed directly on besifloxacin nanosuspension samples, without dilution, in triplicate at 25 ◦C.

#### *2.7. Osmolality*

The osmolality of the optimized formulation was determined using the A2O Advanced Automated Osmometer (Advanced Instruments, Norwood, MA, USA). For this, 1 mL of the nanosuspension was transferred to a glass tube and placed on the disk of the equipment that performs the injection automatically. In this equipment, measurements were determined from the freezing point depression of the sample. The sample injection volume was 100 µL, and the temperature range for determining the freezing point was –40 ◦C to +45 ◦C. Measurements were performed in triplicate.

#### *2.8. Validation of the Method by High-Performance Liquid Chromatography (HPLC)*

The reference method was developed by COSTA and colleagues using the commercial product Besivance® (0.6% ophthalmic suspension) [12]. The following performance characteristics were determined: linearity, selectivity, precision, accuracy, and system suitability [32,33]. The excipients of the commercial product were measured during a validation study (selectivity) in the reference method [12]. Additionally, a placebo was prepared to contain Povacoat® (9 mg/mL), glycerol (10 mg/mL), and benzalkonium chloride (1 mg/mL) to evaluate possible interferences in the analytical responses of this method.

#### 2.8.1. HPLC System and Apparatus

The Dionex™ UltiMate™ 3000 UHPLC+ liquid chromatography (Thermo Fisher Scientific, Waltham, MA, USA) was used, consisting of a DGP-3600 quaternary pump, ACC-3000 autosampler (with sample thermostat), ACC-3000 column compartment, and DAD detector 3000 (RS) equipped with Chromeleon™ software version 6.80. The C18 Eclipse Plus 150 mm × 4.6 mm × 3.5 µm column (Agilent Technologies, Santa Clara, CA, USA) was used. The mobile phase consisted of a mixture of 0.5% (*v*/*v*) triethylamine solution (pH adjusted to 3.0 with dilute phosphoric acid (10% *v*/*v*)) and acetonitrile (74:26 *v*/*v*) at a flow rate of 1.0 mL/min. The injection volume of the solutions was 20 µL, with a column temperature of 25 ◦C, UV detection at 295 nm, using a diode matrix (DAD) [12].

#### 2.8.2. Solutions

Stock solutions of the standard (SQR) and samples containing 500 µg/mL of besifloxacin were prepared. From these solutions, aliquots were diluted using the mobile phase to obtain adequate concentrations to determine the performance characteristics of the method. The solutions were transferred to a 2 mL vial using a Millex®-HV-PVDF filter 13 mm in diameter, 0.45 µm pore size, and then analyzed. Additionally, a filter test was performed to assess the compatibility and possible interference in the analytical responses [34].

The density of the besifloxacin nanosuspension (0.6 wt%) was determined as 1.0081 g/mL. This result was considered for weighing the aliquot of the nanosuspension. Afterwards, a sample solution was prepared with a concentration of 50 µg/mL of besifloxacin.

#### *2.9. Scanning Electron Microscopy (SEM)*

The raw material (besifloxacin hydrochloride) and nanocrystals (lyophilized nanosuspension) were analyzed using a Quanta™ 650 FEG scanning electron microscope (SEM) (FEI Company, Hillsboro, OR, USA). The samples were plated with platinum using a metallizer MED-020 high-vacuum coating system (Baltic, Pfäffikon, Switzerland). This metallization (the layer between 10–15 nm thick) guarantees the conduction of the electron beam in the analyzed sample. For analysis, 10 kV of electron acceleration voltage was applied. The raw material besifloxacin was analyzed at 5000 to 30,000 times magnification, and the nanocrystal was analyzed at 5000 to 100,000 times magnification.

### *2.10. Lyophilization*

The lyophilized samples were used for the analyses that needed dry samples (lyophilized powder), which was obtained by drying the nanosuspension using the

lyophilization process using a Christ Alpha 2-4 LSC (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). For this, 1.5 mL aliquots of nanosuspension were frozen at −45 ◦C with a pressure of 0.120 mbar for 48 h. After lyophilization, the besifloxacin nanocrystals were resuspended in the same volume of pH 7.0 buffer (proportional to the volume of the initial formulation), and the Z-ave, IP, and PZ were determined to verify the efficiency of lyophilization.

#### *2.11. Thermal Analysis*

Differential scanning calorimetry (DSC) analysis was performed using DSC 7020 equipment (Hitachi High-Tech Corporation, Tokyo, Japan). All measurements were performed under a dynamic nitrogen atmosphere (50 mL min−1 ). The weight of the samples was approximately 2 mg. DSC curves were obtained by heating at a rate of 10 ◦C min−1 from 25 to 300 ◦C. As a reference, an empty sealed aluminum crucible was used.

The thermogravimetry and derived thermogravimetry (TG/DTG) curves were obtained using an STA 7200 analyzer (Hitachi High-Tech Corporation, Tokyo, Japan), with a sample weight of approximately 2 mg in the temperature range from 30 to 600 ◦C, at a rate of 10 ◦C min−1 under a dynamic nitrogen atmosphere (100 mL min−1 ). The lyophilized nanosuspension, besifloxacin (raw material), Povacoat®, benzalkonium chloride, and a mixture of both (same proportion of the formulation) were characterized.

#### *2.12. X-ray Diffraction (XRD)*

X-ray diffraction analyses were performed using a Rigaku Miniflex 600-C diffractometer (Rigaku Corporation, Tokyo, Japan). For this, the samples were ground to obtain a fine powder. Next, the powder was spread evenly in the rotating glass sample holder (zero background) with the aid of a glass slide. The equipment was configured in reflection mode with a scan range from 2.3◦ to 50◦ 2Θ, and a speed of 10◦/min (0.02◦ step) was applied. The diffractograms were generated by the SmartLab Studio II software (Rigaku Corporation, Tokyo, Japan).

#### *2.13. Saturation Solubility*

The saturation solubility was evaluated using the shake-flask method [35], using a Tecnal TE 4080 (Tecnal, São Paulo Brazil) stirring oven at 37 ± 1 ◦C with stirring 150 RPM. For the study, the following media were considered: ultrapure water Milli-Q® Integral 10 (Merck KGaA, Darmstadt, Germany), pH 4.0 phosphate buffer (citric acid/sodium hydroxide/hydrogen chloride), Certipur® (Merck KGaA, Darmstadt, Germany), phosphate buffer pH 6.8, and phosphate buffer pH 7.0 (disodium dipotassium hydrogen phosphate hydrogen phosphate) Certipur® (Merck KGaA, Darmstadt, Germany).

To prepare the saturated solution of the drug, 250 mg of besifloxacin (raw material) was weighed and transferred to an Erlenmeyer flask containing 250 mL of the previously described media (concentration 1 mg/mL). Similarly, 4130 mg of the besifloxacin nanosuspension (equivalent to 25 mg of besifloxacin, density 1.0081 g/mL) was transferred to Erlenmeyer of 25 mL containing 25 mL of the media (concentration 1 mg/mL). For these preparations, the declared solubility of besifloxacin of 0.143 mg/mL was considered [5].

The samples were incubated, and after 24 and 48 h, an aliquot of 1 mL was removed, filtered Millex®-HV-PVDF 13 mm in diameter, with a pore size of 0.45 µm. From this filtrate, solutions with a theoretical concentration of 50 µg/mL were prepared using the respective media (ultrapure water, pH 4.0 buffer, pH 6.8 buffer, and pH 7.0 buffer). Afterwards, the sample solutions were transferred to a 2 mL vial using Millex®-HV-PVDF filters 13 mm in diameter, pore size 0.45 µm, and quantified in triplicate (three injections) by HPLC.

#### *2.14. In Vivo Toxicity*

Larvae of *Galleria mellonella* ranging from 2.0 to 2.5 cm in length and 150 to 200 mg in weight were used to determine the toxicity of besifloxacin nanocrystals and the Povacoat®. Doses of 500 mg/kg of besifloxacin nanocrystals (0.06 mg/larvae) and 750 mg/kg of

Povacoat® (0,09 mg/larvae) were injected (10 µL) in the last left pro-leg of larvae (*n* = 16 larvae per group). In addition, sterile phosphate-buffered saline (pH 7.0) was also injected into a larval group to control mechanical injury and vehicle. The larvae were maintained at 37 ◦C, and the survival of all groups was monitored daily for 5 days [36].

#### *2.15. Determination of Minimum Inhibitory Concentration (MIC)*

The minimum inhibitory concentration (MIC) was determined for besifloxacin hydrochloride (raw material) and for besifloxacin nanosuspension 0.6 wt% against *Staphylococcus aureus* and *Pseudomonas aeruginosa*. Similarly, a serial dilution method was used, according to item 2.2.2 [29].

#### *2.16. Stability Study*

The accelerated stability study of the besifloxacin nanosuspension was conducted in a Climacell Eco-Line climate chamber (MMM Medcenter Einrichttungen GmbH, Planegg, Germany) considering the main regulatory guidelines [37,38]. For this purpose, three batches of the formulation were prepared, and the samples were transferred to type I amber glass vials, closed with butyl caps, sealed with an aluminum seal, and conditioned for accelerated stability for 90 days at the condition: 40 ◦C ± 2 ◦C/75%RH ± 5%RH.

Monthly, Z-ave, PdI, ZP, pH, and aspect were determined. In addition, the besifloxacin content was determined at time 0 and after 90 days. Analysis of variance (ANOVA) was performed using Excel software.

#### **3. Results and Discussion**

#### *3.1. Drug Analysis*

rial conjunctivitis.

3.1.1. Particle Size Determination by Laser Diffraction

The initial particle size distribution for besifloxacin hydrochloride (raw material) and commercial product (Besivance® 0.6% ophthalmic suspension) was determined as a preliminary analysis. The particle size distribution (Figure 1) shows the same bimodal formation for both product and raw material samples. The central peak presents the most extensive distribution of particles, with a diameter 9.33 ± 3.22 µm and 10.62 ± 1.76 µm for raw material and Besivance®, respectively. In addition, the D90 shows that 90% of the particles have a diameter smaller than 13 µm for both samples. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 8 of 25 Therefore, the raw material besifloxacin hydrochloride is presented on a micrometric scale, close to the reference values (commercial product). Drug nanocrystal prepared using small-scale wet bead milling can overcome the low water solubility hurdle of the raw material, which potentially improves drug bioavailability justifying the present study.

Likewise, the in vitro antimicrobial activity of besifloxacin hydrochloride (raw material) and Besivance® were determined using microorganisms that frequently cause bacte-

MIC for *Pseudomonas aeruginosa* against besifloxacin hydrochloride and the product Besivance® was 1.58 and 0.99 µg/mL, respectively. The values reported in the literature are between 0.5 and 4 µg/mL [8,29,39]. Against *Staphylococcus aureus*, the MIC was 0.0957 and 0.0959 µg/mL for besifloxacin hydrochloride and the product Besivance®, respectively. The literature reported values between 0.008 and 8 µg/mL [8,29,39]. Thus, the raw material met the requirements for use in the drug nanocrystal development process.

The choice of stabilizer is complex and critical in the development stage of nanocrystals. In the present study, 11 stabilizers were tested to obtain nanocrystals. These stabilizers were compared considering their efficiency in particle size reduction and the preliminary results for their physical stability. For this, 24 formulations were evaluated with

In the first exploratory study, the stabilizers conventionally used to obtain nanocrystals were tested (Figure S1), and no significant size reduction or no physical stability was observed. In the second part, formulations with new and promising stabilizers were prepared (Figure S2). Therefore, a study was carried out to evaluate the stability of the formulations with the stabilizers that reduced the size of the particles to the nanometric scale

From the exploratory study, it was found that the stabilizer Povacoat® allowed us to obtain an innovative formulation for treating bacterial conjunctivitis. Figure 2 shows formulas F1, F3, F7, and F8 selected from preliminary studies. However, after 15 days, F1

a single stabilizer or combinations, divided into three stages.

in the first and second exploratory studies (Table S1).

Applying Box–Behnken Experiment Design

3.1.2. Determination of Minimum Inhibitory Concentration (MIC)

*3.2. Preparation of Besifloxacin Nanocrystals Using Small-Scale Wet Bead Milling*  3.2.1. Exploratory Tests for Stabilizer and Nanosuspension Optimization Process

Therefore, the raw material besifloxacin hydrochloride is presented on a micrometric scale, close to the reference values (commercial product). Drug nanocrystal prepared using small-scale wet bead milling can overcome the low water solubility hurdle of the raw material, which potentially improves drug bioavailability justifying the present study.

#### 3.1.2. Determination of Minimum Inhibitory Concentration (MIC)

Likewise, the in vitro antimicrobial activity of besifloxacin hydrochloride (raw material) and Besivance® were determined using microorganisms that frequently cause bacterial conjunctivitis.

MIC for *Pseudomonas aeruginosa* against besifloxacin hydrochloride and the product Besivance® was 1.58 and 0.99 µg/mL, respectively. The values reported in the literature are between 0.5 and 4 µg/mL [8,29,39]. Against *Staphylococcus aureus*, the MIC was 0.0957 and 0.0959 µg/mL for besifloxacin hydrochloride and the product Besivance®, respectively. The literature reported values between 0.008 and 8 µg/mL [8,29,39]. Thus, the raw material met the requirements for use in the drug nanocrystal development process.

#### *3.2. Preparation of Besifloxacin Nanocrystals Using Small-Scale Wet Bead Milling*

3.2.1. Exploratory Tests for Stabilizer and Nanosuspension Optimization Process Applying Box–Behnken Experiment Design

The choice of stabilizer is complex and critical in the development stage of nanocrystals. In the present study, 11 stabilizers were tested to obtain nanocrystals. These stabilizers were compared considering their efficiency in particle size reduction and the preliminary results for their physical stability. For this, 24 formulations were evaluated with a single stabilizer or combinations, divided into three stages.

In the first exploratory study, the stabilizers conventionally used to obtain nanocrystals were tested (Figure S1), and no significant size reduction or no physical stability was observed. In the second part, formulations with new and promising stabilizers were prepared (Figure S2). Therefore, a study was carried out to evaluate the stability of the formulations with the stabilizers that reduced the size of the particles to the nanometric scale in the first and second exploratory studies (Table S1).

From the exploratory study, it was found that the stabilizer Povacoat® allowed us to obtain an innovative formulation for treating bacterial conjunctivitis. Figure 2 shows formulas F1, F3, F7, and F8 selected from preliminary studies. However, after 15 days, F1 containing Povacoat® presented physical stability. Developed as a pharmaceutical excipient, Povacoat®, with consolidated use for coating and wet granulation processes, has a pKa of 3.56 in strong acid and 9.5 in strong base, with a load of 0.03 in pH 6.5. This copolymer is established as a stabilizing agent for obtaining nanocrystals, preventing aggregations, and improving the saturation solubility of nanosuspensions with drugs with nanometer-scale particles [28,40–46].

The concept of design space has been consolidated as a fundamental step in pharmaceutical development. This approach allows the understanding and establishing multivariate combinations and interactions of input variables for the proper development of the product or process [28,47,48]. The main variables found in exploratory studies were considered in this optimization.

The concentration of Povacoat® (1.0 wt%) obtained in the preliminary investigation was considered to establish the maximum concentration of this stabilizer for the experimental design (+1). The central level (0) was 0.8 wt% and the minimum concentration (−1) established was 0.6 wt%, with the prospect of obtaining the same concentration concerning the drug (1:1). This concentration was also tested in preliminary trials. Although nonionic stabilizers are less toxic than cationic, these should be used with as little as possible concentration to avoid patient risks [48].

nometer-scale particles [28,40–46].

containing Povacoat® presented physical stability. Developed as a pharmaceutical excipient, Povacoat®, with consolidated use for coating and wet granulation processes, has a pKa of 3.56 in strong acid and 9.5 in strong base, with a load of 0.03 in pH 6.5. This copol-

**Figure 2.** Aspect of the formulas containing the stabilizers Povacoat® 1.0 wt% (F1), Kollicoat® 0.3 wt% + Chitosan 0.3 wt% (F3), Poloxamer® 407 0.5 wt% + Chitosan 0.5 wt% (F7), and Poloxamer® 407 0.3 wt% + Chitosan 0.3 wt% (F8). Stability after 15 days (25 °C). **Figure 2.** Aspect of the formulas containing the stabilizers Povacoat® 1.0 wt% (F1), Kollicoat® 0.3 wt% + Chitosan 0.3 wt% (F3), Poloxamer® 407 0.5 wt% + Chitosan 0.5 wt% (F7), and Poloxamer® 407 0.3 wt% + Chitosan 0.3 wt% (F8). Stability after 15 days (25 ◦C).

The concept of design space has been consolidated as a fundamental step in pharmaceutical development. This approach allows the understanding and establishing multivariate combinations and interactions of input variables for the proper development of the product or process [28,47,48]. The main variables found in exploratory studies were considered in this optimization. The concentration of Povacoat® (1.0 wt%) obtained in the preliminary investigation For drug substance concentration, the following levels were considered: (−1), central (0) and (+1), 0.4, 0.6, and 0.8 (wt%), respectively, aiming to evaluate a possible synergistic effect in the reduction of Z-ave. Speed is also a critical parameter of the process; therefore, in the experiment (−1), central (0), and (+1) were 800, 1000, and 1200 rpm, respectively. The measurement of Z-ave was taken after 24 h of preparation (Table 2).

was considered to establish the maximum concentration of this stabilizer for the experimental design (+1). The central level (0) was 0.8 wt% and the minimum concentration (−1) established was 0.6 wt%, with the prospect of obtaining the same concentration concerning the drug (1:1). This concentration was also tested in preliminary trials. Although **Table 2.** Box–Behnken experimental design and observed responses. Average hydrodynamic diameter (Z-ave) in nm was obtained for the besifloxacin nanocrystal preparations. Conditions: 30% (m/m) zirconium beads and 2 magnetic bars, 25 ◦C.

According to Table 2, it is possible to observe that the maximum and minimum values of Z-ave were 748.4 ± 47.6 nm and 578.9 ± 12.3 nm, respectively, and that IP values were less than 0.2. For this optimization, the analysis of variance (Table 3) showed that the quadratic model was significant (*p*-value less than 0.05; α < 0.05) and presented a nonsignificant value for lack of fit (*p*-value = 0.154). The variables showed significant quadratic terms: besifloxacin\*besifloxacin, Povacoat®\*Povacoat®, speed\*speed. In addition, interactions between besifloxacin and Povacoat® and besifloxacin and rate were observed. The interaction between besifloxacin and Povacoat® reduced particle size, while the interaction

between besifloxacin and velocity favored its increase (Equation (1)). Additionally, the quadratic terms influence the Z-ave (increasing). These impacts are also shown in the Z-ave contour plots (Figure 3).

**Table 3.** Analysis of variance to test the significance of the terms obtained for the particle Z-ave variation of the besifloxacin nanocrystal preparations. The variables are besifloxacin concentration (wt%), Povacoat® concentration (wt%), and velocity (rpm).

DF: degree of freedom; Adj. SS: Sum of the adjusted squares; Adj. MS: Adjusted quadratic mean, F-value: F statistic; *p*-value: significance level; (\*): not calculated. Besifloxacin square (Besifloxacin \* Besifloxacin); Povacoat®square (Povacoat®\* Povacoat®); Speed square (Speed \* Speed); interaction between Besifloxacin and Povacoat®(Besifloxacin \* Povacoat®), and interaction between Besifloxacin and Speed (Besifloxacin \* Speed). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 12 of 25

**Figure 3.** Contour graph related to the Z-ave evaluation of the besifloxacin nanocrystal, whose variables are besifloxacin concentration (wt%), Povacoat® concentration (wt%), and velocity (rpm). **Figure 3.** Contour graph related to the Z-ave evaluation of the besifloxacin nanocrystal, whose variables are besifloxacin concentration (wt%), Povacoat® concentration (wt%), and velocity (rpm).

The regression equation obtained for the mathematical model of the experiment is

(1)

<math> <semantics> <mrow> <mtext>Z</mtext><mo>−</mo><mtext>ave</mtext><mo>=</mo><mn>1608</mn><mo>−</ mo><mn>637</mn><mtext> Besifloxacin</mtext><mo>−</mo><mn>689</mn><mtext> Povacoat®</mtext><mo>−</mo><mn>1.021</mn><mtext> Speed</mtext><mo>+</mo ><mn>1100</mn><mtext> Besifloxacin\*Besifloxacin</mtext><mo>+</mo><mn>790</mn><mtext> Povacoat®\*Povacoat®</mtext><mo>+</mo><mn>0.000383</mn><mtext> Speed\*Speed</mtext><mo>&#x 2212;</mo><mn>1200</mn><mtext> Besifloxacin\*Povacoat®</mtext><mo>+</mo><mn>0.370</mn><mtext> Besifloxacin\*Speed</mtext><mo>.</mo></mrow> <annotation encoding='MathType-MTEF'>MathType@MTEF@5@5@+= feaagKart1ev2aqatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr

described in Equation (1).

The regression equation obtained for the mathematical model of the experiment is described in Equation (1).

Z − ave = 1608 − 637 Besifloxacin − 689 Povacoat − 1.021 Speed + 1100 Besifloxacin ∗ Besifloxacin + 790 Povacoat ∗ Povacoat + 0.000383 Speed ∗ Speed − 1200 Besifloxacin ∗ Povacoat + 0.370 Besifloxacin ∗ Speed. (1)

where Z-ave: average hydrodynamic diameter.

The coefficient of determination and the adjusted coefficient of determination (R2 = 98.37% and R2 (Adj.) = 96.16%) are close, allowing the conclusion that nonsignificant values were not considered for the model (Table 4). The predicted R-squared indicates how well a regression model predicts responses to new observations. In this case, the R2 (pred) was 75.85%. Generally, values above 70% indicate an excellent forecast.

**Table 4.** Significance of coded regression coefficients and fit index of the selected model in evaluating Z-ave nanocrystals of besifloxacin.

Coef: Coefficient; SE Coef: Standard deviation coefficient; *t*-value: size of the difference relative to the variation; *p*-value: level of significance of terms; VIF: variance inflation factor; S: standard deviation; R2 : coefficient of determination; R2 (adj.): Adjusted coefficient of determination; R2 (pred): Coefficient of prediction determination of the adjusted model. Besifloxacin square (Besifloxacin \* Besifloxacin); Povacoat®square (Povacoat®\* Povacoat®); Speed square (Speed \* Speed); interaction between Besifloxacin and Povacoat®(Besifloxacin \* Povacoat®), and interaction between Besifloxacin and Speed (Besifloxacin \* Speed).

### 3.2.2. Mathematical Model Verification

Two formulations with target particle sizes were prepared (F1: target 600 nm, F2: target 576 nm). The same concentration of the reference product was used in this study (0.6% m/m), which is essential to guarantee the minimum inhibitory concentration (MIC). For the formulations, the same conditions were maintained: 30% zirconium spheres and two magnetic bars, with the total amount adjusted to 10 g with phosphate buffer (pH 7.0). The experimental Z-ave (F1 and F2) are close to the theoretical values of each formulation. Therefore, they show the validity of the model (Table 5). Thus, the besifloxacin nanosuspension 0.6 wt% (F2) was selected for posterior analysis due to its reduced particle size (560.3 ± 4.2 nm).

**Table 5.** Variables obtained in the Box–Behnken experimental design for the optimized nanocrystal preparations and respective theoretical and experimental Z-ave values.

#### *3.3. Distribution and Particle Size by Laser Diffraction (LD)* properties or phenomena, including physical, chemical, or biological effects that are

*3.3. Distribution and Particle Size by Laser Diffraction (LD)*

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 15 of 25

Nanotechnology-based materials or final products are those designed to exhibit properties or phenomena, including physical, chemical, or biological effects that are attributable to its dimensions, even if they are smaller than 1 µm (1000 nm) in size [49]. attributable to its dimensions, even if they are smaller than 1 µm (1000 nm) in size [49]. Given the importance of particle size in nanosuspensions, one of the main characteristics of nanoscale products [50], it is essential to verify particle size by two

Nanotechnology-based materials or final products are those designed to exhibit

Given the importance of particle size in nanosuspensions, one of the main characteristics of nanoscale products [50], it is essential to verify particle size by two different techniques. Therefore, a new batch of besifloxacin nanosuspension 0.6 wt% was prepared and analyzed by laser diffraction (LD) and dynamic light scattering (DLS), the latter being the primary technique used during this study. The determination by DLS is related to the Brownian motion of the particles, which promotes the scattering of laser light at different intensities. Consequently, the particle size was determined using the Stokes–Einstein relationship. The particle size distribution by LD is related to the modification of the scattered light intensity as a function of the scattering angle, based on Mie's theory [31,51]. different techniques. Therefore, a new batch of besifloxacin nanosuspension 0.6 wt% was prepared and analyzed by laser diffraction (LD) and dynamic light scattering (DLS), the latter being the primary technique used during this study. The determination by DLS is related to the Brownian motion of the particles, which promotes the scattering of laser light at different intensities. Consequently, the particle size was determined using the Stokes–Einstein relationship. The particle size distribution by LD is related to the modification of the scattered light intensity as a function of the scattering angle, based on Mie's theory [31,51]. A saturated drug solution was used for dispersion in the DLS technique and

A saturated drug solution was used for dispersion in the DLS technique and phosphate buffer (pH 7.0) for the LD technique. These precautions are essential to maintain the physical characteristics of the nanocrystals during measurements [42]. phosphate buffer (pH 7.0) for the LD technique. These precautions are essential to maintain the physical characteristics of the nanocrystals during measurements [42]. The DLS analysis showed Z-ave = 531.1 ± 2 .7 nm and PdI = 0.102 ± 0.031 (Figure S3),

The DLS analysis showed Z-ave = 531.1 ± 2 .7 nm and PdI = 0.102 ± 0.031 (Figure S3), while the LD analysis showed D90 = 0.309 ± 0.003 µm, where 95.08% of the particles have a size ≤0.564 ± 0.005 µm, as shown in Figure 4. Thus, the nanosuspension of besifloxacin 0.6% wt% has a particle size approximately 17 times smaller compared to the average particle size of besifloxacin hydrochloride (raw material) and the commercial product (Besivance®) (approximately 10 µm). Furthermore, the similarity between the results of the two techniques (DLS and DL) showed that the milling process was adequate. while the LD analysis showed D90 = 0.309 ± 0.003 µm, where 95.08% of the particles have a size ≤0.564 ± 0.005 µm, as shown in Figure 4. Thus, the nanosuspension of besifloxacin 0.6% wt% has a particle size approximately 17 times smaller compared to the average particle size of besifloxacin hydrochloride (raw material) and the commercial product (Besivance®) (approximately 10 µm). Furthermore, the similarity between the results of the two techniques (DLS and DL) showed that the milling process was adequate.

**Figure 4.** Particle size distribution of besifloxacin nanosuspension 0.6 wt% by laser diffraction (LD). Representative of three runs (*n* = 3). **Figure 4.** Particle size distribution of besifloxacin nanosuspension 0.6 wt% by laser diffraction (LD). Representative of three runs (*n* = 3).

#### *3.4. pH, Density, and Viscosity 3.4. pH, Density, and Viscosity*

The pH for the optimized formulation (besifloxacin nanosuspension 0.6 wt%) was 6.52 using phosphate buffer (pH = 7.0) as diluent. In this condition, the drug is not solubilized, unlike the manufacturing process of the reference product (Besivance®). In this case, besifloxacin hydrochloride is dissolved, and after dissolution, the drug is precipitated at pH 6.5 (zwitterion form of the active) [52]. Thus, more steps are required to obtain the commercial product than the besifloxacin nanosuspension. The pH for the optimized formulation (besifloxacin nanosuspension 0.6 wt%) was 6.52 using phosphate buffer (pH = 7.0) as diluent. In this condition, the drug is not solubilized, unlike the manufacturing process of the reference product (Besivance®). In this case,besifloxacin hydrochloride is dissolved, and after dissolution, the drug is precipitated at pH 6.5 (zwitterion form of the active) [52]. Thus, more steps are required to obtain the commercial product than the besifloxacin nanosuspension.

Therefore, this pH value (pH = 6.5) is within the reference of the United States Pharmacopoeia [53], which establishes that pH between 3.0 and 8.6 is tolerable for ophthalmic formulations. Furthermore, this result can be considered ideal without causing discomfort to patients since tear pH ranges from 6.5 to 7.6, with a mean value of Therefore, this pH value (pH = 6.5) is within the reference of the United States Pharmacopoeia [53], which establishes that pH between 3.0 and 8.6 is tolerable for ophthalmic formulations. Furthermore, this result can be considered ideal without causing discomfort to patients since tear pH ranges from 6.5 to 7.6, with a mean value of 7.0 [54]. Still, the nanosuspension is suitably buffered, avoiding pH fluctuations and contributing to its stability.

The density of nanosuspension was 1.0081 g/mL, with an average viscosity of 2.40 ± 0.01 cP. These values are within the range of tear fluid viscosity, varying from 1.05 to 5.97 cP. In addition, preparations with a viscosity below 20 cP do not cause discomfort to patients [55]. Determining the viscosity of ophthalmic preparations is not a compendial requirement but must be considered for product specifications, and depends on the proposed formulation [53].

Adequate viscosity allows better Z-ave stability, avoiding aggregation/agglomeration. The Povacoat® is likely adsorbed on the nanocrystal surface, reducing the risk of agglomeration of nanoparticles related to the Ostwald ripening phenomenon [22,56]. In addition, the increase in viscosity favors an increase in the residence time of the drug in the eye, improving bioavailability. However, high viscosity can inhibit the diffusion of drugs from the formulation to the eye [53]. Additionally, high viscosity can increase patient discomfort (pain and blurred vision) and result in dose variability [17]. In the case of the commercial product (Besivance®), DuraSite™ technology offers a viscosity of approximately 1500 cps [14].

### *3.5. Osmolality*

The mean osmolality obtained for the besifloxacin nanosuspension (0.6 wt%) was 164.3 ± 1.5 mOsmol/kg. The United States Pharmacopeia establishes that the default value for the isosmotic solution is 0.9 wt% sodium chloride (NaCl) solution, which has an osmolality of 285 mOsmol/kg and an osmolarity of 284 mOsmol/L [57]. Therefore, it was proposed to adjust the osmolality of the nanosuspension of besifloxacin 0.6 wt% (optimized formula). Usually, osmolality adjustment can be performed with 0.9 wt% sodium chloride solution; however, this is not recommended for the adjustment of nanosuspensions, as it can change the zeta potential and destabilize the formulation. For this, glycerol was considered, which is a neutral osmotic agent [58].

After adding 1.0 wt% of glycerol, an average osmolality of 303.7 ± 0.6 mOsmol/kg was obtained, close to the typical values of 0.9% sodium chloride solution, as recommended by the United States Pharmacopoeia. Values between 171 and 1711 mOsmol/kg can be tolerated without discomfort to the patient [57]. In addition, these values are like those declared for the reference product Besivance® (290 mOsmol/kg) [5,14].

#### *3.6. Validation of the Method by High Performance Liquid Chromatography (HPLC)*

For specificity, a placebo containing Povacoat® (stabilizer), glycerol (isotonizing agent), and benzalkonium chloride (preservative), used in the optimized formulation, did not interfere with the analytical responses. In addition, a test was performed using Millex®- HV-PVDF filters 13 mm in diameter, with 0.45 µm pores, which had no influence on the analytical responses from the filtering of the solutions.

The method was linear, presenting a correlation coefficient of r = 0.9996 (R2 = 99.96%) for drug concentrations ranging from 20 to 80 µg/mL. In the precision by repeatability (intraday), the following values of relative standard deviation (RSD) were obtained: 1.20% on the first day, 0.84% on the second day, and 0.39% on the third day. Regarding intermediate precision, the analyses provided an RSD of 0.94%. The accuracy was evaluated by the recovery method (addition of the standard) and was shown to be satisfactory (99.03%). Therefore, the method is adequate, like the one validated by COSTA and colleagues [12], meeting the recommendations of the ICH guide and United States Pharmacopeia [32,33].

#### *3.7. Scanning Electron Microscopy (SEM)*

Scanning electron microscopy (SEM) showed the morphology of the raw material besifloxacin (Figure 5a) and the besifloxacin nanocrystals (Figure 5b). Microphotographs showed a change from the irregular prismatic crystal to an approximately spherical shape after the milling process. Furthermore, the micrographs showed that the nanocrystals are involved in a light layer, likely related to the Povacoat® stabilizer [42].

involved in a light layer, likely related to the Povacoat® stabilizer [42].

Scanning electron microscopy (SEM) showed the morphology of the raw material besifloxacin (Figure 5a) and the besifloxacin nanocrystals (Figure 5b). Microphotographs showed a change from the irregular prismatic crystal to an approximately spherical shape after the milling process. Furthermore, the micrographs showed that the nanocrystals are

**Figure 5.** Microphotography of raw material besifloxacin (**a**) and besifloxacin nanocrystals (**b**). **Figure 5.** Microphotography of raw material besifloxacin (**a**) and besifloxacin nanocrystals (**b**).

#### *3.8. Lyophilization 3.8. Lyophilization*

The lyophilized samples were used for the analyses that needed dry samples. The samples before lyophilization showed Z-ave = 510.5 ± 8.5 nm, PdI = 0.052 ± 0.060 and PZ = −9.66 ± 0.27. After the drying process, these characteristics did not change, presenting Zave = 514.4 ± 27.0 nm, PdI = 0.153 ± 0.118, and PZ = −8.66 ± 0.68. Therefore, the lyophilization method was adequate and did not change the characteristics of the nanocrystals. In addition, we verified that the redispersion time of the nanocrystals was fast, and no apparent aggregates were observed. Unlike other lyophilization processes [42], the besifloxacin nanocrystals were successfully dried without adding a cryoprotectant substance. Avoiding the need to add substances is essential due to the restriction of using substances in ophthalmic preparations [59]. The lyophilized samples were used for the analyses that needed dry samples. The samples before lyophilization showed Z-ave = 510.5 ± 8.5 nm, PdI = 0.052 ± 0.060 and PZ = −9.66 ± 0.27. After the drying process, these characteristics did not change, presenting Z-ave = 514.4 ± 27.0 nm, PdI = 0.153 ± 0.118, and PZ = −8.66 ± 0.68. Therefore, the lyophilization method was adequate and did not change the characteristics of the nanocrystals. In addition, we verified that the redispersion time of the nanocrystals was fast, and no apparent aggregates were observed. Unlike other lyophilization processes [42], the besifloxacin nanocrystals were successfully dried without adding a cryoprotectant substance. Avoiding the need to add substances is essential due to the restriction of using substances in ophthalmic preparations [59].

#### *3.9. Thermal Analysis 3.9. Thermal Analysis*

The differential scanning calorimetry (DSC) (Figure 6) showed the expected thermal temperature profile for each formulation component analyzed individually. Regarding besifloxacin (raw material), no thermal events were observed in the temperature range studied, consistent with Santos and colleagues [17]. The differential scanning calorimetry (DSC) (Figure 6) showed the expected thermal temperature profile for each formulation component analyzed individually. Regarding besifloxacin (raw material), no thermal events were observed in the temperature range studied, consistent with Santos and colleagues [17]. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 18 of 25

**Figure 6.** DSC curves of formulation components, mixture (same proportion as formulation), and besifloxacin nanocrystals. **Figure 6.** DSC curves of formulation components, mixture (same proportion as formulation), and besifloxacin nanocrystals.

individual components, mainly Povacoat® (Figure 6). Therefore, the analysis showed the thermal stability of the nanocrystals, since no degradation events were observed up to 180 °C. Furthermore, the similarity of the DSC curves of the nanocrystals and the mixture shows that the crystalline form was probably maintained. Additionally, this guarantees

For the nanocrystals, an endothermic curve was observed at approximately 180 °C;

According to the XRD obtained for the nanocrystals (Figure 7), it is possible to observe the prominent peaks of besifloxacin in the 2Θ angles of 10.6, 19.7, and 21.1°, revealing that the crystalline form is likely predominant in the lyophilized formulation. The besifloxacin peaks are also shown in Figure S4. Similar results were presented by Harry [60], where besifloxacin hydrochloride is characterized by diffractograms with

Regarding the peaks formed after the angle 2Θ 30°, these are mainly related to the free base of besifloxacin, which present low solubility at pH 6.5, condition of the milling process, and nanosuspension. The same characteristics were observed during the manufacturing process of the Besivance® product, where the HCl salt is converted into the free base [60]. Still, these peaks may be related to the interaction of the drug's free base with the Povacoat® stabilizer (Figure 7) for the formation of the nanocrystal. Therefore,

peaks at 2Θ angles of 10.6, 15, 19.7, 21.1, and 22° ± 0.2.

the crystalline phase is possibly predominant in the nanosuspension.

the compatibility of the components.

*3.10. X-ray Diffraction (XRD)*

For the nanocrystals, an endothermic curve was observed at approximately 180 ◦C; in this case, it shifted at a previous temperature with the mixture (197 ◦C). However, it is possible to show that this curve is related to the sum of the thermal events of the individual components, mainly Povacoat® (Figure 6). Therefore, the analysis showed the thermal stability of the nanocrystals, since no degradation events were observed up to 180 ◦C. Furthermore, the similarity of the DSC curves of the nanocrystals and the mixture shows that the crystalline form was probably maintained. Additionally, this guarantees the compatibility of the components.

### *3.10. X-ray Diffraction (XRD)*

According to the XRD obtained for the nanocrystals (Figure 7), it is possible to observe the prominent peaks of besifloxacin in the 2Θ angles of 10.6, 19.7, and 21.1◦ , revealing that the crystalline form is likely predominant in the lyophilized formulation. The besifloxacin peaks are also shown in Figure S4. Similar results were presented by Harry [60], where besifloxacin hydrochloride is characterized by diffractograms with peaks at 2Θ angles of 10.6, 15, 19.7, 21.1, and 22◦ ± 0.2. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 19 of 25

**Figure 7.** X-ray diffraction of besifloxacin (raw material), nanocrystals, mixture (besifloxacin and Povacoat®), and Povacoat®. *n* = 3. **Figure 7.** X-ray diffraction of besifloxacin (raw material), nanocrystals, mixture (besifloxacin and Povacoat®), and Povacoat®. *n* = 3.

*3.11. Saturation Solubility*  Analyses to determine the saturation solubility were conducted under 4 pH conditions (ultrapure water, phosphate buffer pH 4.0, pH 6.0, and pH 7.0) [35]. The concentration of besifloxacin dissolved in each medium was determined by HPLC, using the validated method for quantification and indicative of stability. The tests were performed in three replicates with a theoretical concentration of 50 µg/mL for each condition and showed a relative standard deviation below 1.5% for the media, meeting Regarding the peaks formed after the angle 2Θ 30◦ , these are mainly related to the free base of besifloxacin, which present low solubility at pH 6.5, condition of the milling process, and nanosuspension. The same characteristics were observed during the manufacturing process of the Besivance® product, where the HCl salt is converted into the free base [60]. Still, these peaks may be related to the interaction of the drug's free base with the Povacoat® stabilizer (Figure 7) for the formation of the nanocrystal. Therefore, the crystalline phase is possibly predominant in the nanosuspension.

Figure 8 shows the saturation solubility of besifloxacin hydrochloride (raw material) and nanosuspension. Regarding the incubation time, no significant changes were

increases approximately twice in phosphate buffer at pH 6.8 and 7.0, relative to the raw material. This performance allows to increase the bioavailability of the drug substance [21–24,42]. The decrease in the particle radius with a consequent increase in the curvature of their surface causing an increase in the dissolution pressure, according to the Ostwald-Freundlich equation [25]. The reference product (Besivance®) presents the same pH range [52]. Furthermore, this is the ideal pH range for ophthalmic formulations, considering the

the Brazilian regulatory requirement [35].

tear pH [54].

#### *3.11. Saturation Solubility* Analyses to determine the saturation solubility were conducted under 4 pH conditions (ultrapure water, phosphate buffer pH 4.0, pH 6.0, and pH 7.0) [35]. The concentra-

Povacoat®), and Povacoat®. *n* = 3.

*3.11. Saturation Solubility*

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 18 of 24

Analyses to determine the saturation solubility were conducted under 4 pH conditions (ultrapure water, phosphate buffer pH 4.0, pH 6.0, and pH 7.0) [35]. The concentration of besifloxacin dissolved in each medium was determined by HPLC, using the validated method for quantification and indicative of stability. The tests were performed in three replicates with a theoretical concentration of 50 µg/mL for each condition and showed a relative standard deviation below 1.5% for the media, meeting the Brazilian regulatory requirement [35]. tion of besifloxacin dissolved in each medium was determined by HPLC, using the validated method for quantification and indicative of stability. The tests were performed in three replicates with a theoretical concentration of 50 µg/mL for each condition and showed a relative standard deviation below 1.5% for the media, meeting the Brazilian regulatory requirement [35]. Figure 8 shows the saturation solubility of besifloxacin hydrochloride (raw material) and nanosuspension. Regarding the incubation time, no significant changes were ob-

**Figure 7.** X-ray diffraction of besifloxacin (raw material), nanocrystals, mixture (besifloxacin and

Figure 8 shows the saturation solubility of besifloxacin hydrochloride (raw material) and nanosuspension. Regarding the incubation time, no significant changes were observed after 24 h (Figure 8a) and 48 h (Figure 8b). For nanocrystals, the solubility increases approximately twice in phosphate buffer at pH 6.8 and 7.0, relative to the raw material. This performance allows to increase the bioavailability of the drug substance [21–24,42]. The decrease in the particle radius with a consequent increase in the curvature of their surface causing an increase in the dissolution pressure, according to the Ostwald-Freundlich equation [25]. The reference product (Besivance®) presents the same pH range [52]. Furthermore, this is the ideal pH range for ophthalmic formulations, considering the tear pH [54]. served after 24 h (Figure 8a) and 48 h (Figure 8b). For nanocrystals, the solubility increases approximately twice in phosphate buffer at pH 6.8 and 7.0, relative to the raw material. This performance allows to increase the bioavailability of the drug substance [21–24,42]. The decrease in the particle radius with a consequent increase in the curvature of their surface causing an increase in the dissolution pressure, according to the Ostwald-Freundlich equation [25]. The reference product (Besivance®) presents the same pH range [52]. Furthermore, this is the ideal pH range for ophthalmic formulations, considering the tear pH [54].

**Figure 8.** Saturation solubility of besifloxacin (raw material) and nanosuspension (besifloxacin 0.6 wt%). (**a**) Solubility after 24 h of incubation. (**b**) Solubility after 48 h of incubation. (**c**) pH of the samples at the end of the incubation. Theoretical concentration: 50 µg/mL. *n* = 3.

The solubility of the raw material in water follows the literature (0.143 mg/mL) [5]. However, the nanosuspension showed lower solubility in this medium (11.11 ± 0.43 µg/mL). This is due to the increase in pH to neutrality when the nanosuspension (buffered at pH 7.0) is added to this medium at the beginning of the tests (Figure 8c). In buffer pH 4.0, raw material and nanosuspension showed solubility 50.65 ± 0.94 µg/mL and 51.50 ± 0.19 µg/mL, respectively.

### *3.12. Determination of Minimum Inhibitory Concentration (MIC)*

The MIC values were determined as a function of the presence/absence of microbial growth for the test tubes containing the serial dilutions (0.125 to 60 µg/mL). The MIC for besifloxacin hydrochloride (raw material) and the nanosuspension against *Pseudomonas aeruginosa* was 1.60 µg/mL. Against *Staphylococcus aureus*, the MIC for besifloxacin (raw material) and the nanosuspension were 0.0958 and 0.0960 µg/mL, respectively. These values are like those observed in the preliminary analyses of the raw material and the product (item 3.3). Furthermore, they agree with the literature [8,29,39]. Thus, the milling process did not interfere with the in vitro antibiotic efficacy. Similar results were reported for different nano-based besifloxacin delivery: liposome [17], nanoemulsion [18], nanostructured lipid carrier [19], and nanofibrous [20] (Table S2).

#### *3.13. In Vivo Toxicity*

Larvae of *Galleria mellonella* (*n* = 16) were injected using a maximal dose of besifloxacin nanosuspension, Povacoat®, and the phosphate buffer (vehicle control). Besifloxacin nanosuspension (500 mg/kg) and the Povacoat® (750 mg/kg) did not show toxicity (Figure S5). The three groups presented 100% larvae survival at the end of the experiment (5 days). An in vivo toxicity study in this model has been used [36,61], mainly because conventional in vitro methods show limitations that make it difficult to simulate physiological responses [62]. Furthermore, this method can support preliminary decisions, reducing the number of tests in mammals [63].

#### *3.14. Stability Study*

An accelerated stability study was carried out in order to evaluate possible physicochemical changes in the nanosuspension, and samples were incubated at 40 ◦C ± 2 ◦C/75% RH ± 5% RH. As for the aspect, evaluation of the samples carried out at each time point of the study indicate homogeneity, without change in color, odor, phase separation, and significant sample sedimentation. At each moment, slight sediment of approximately 1 mm in thickness was seen at the bottom of the flasks; this sediment is broken up easily through manual homogenization.

About Z-ave, an increase above 50 nm was observed between the initial analysis (time 0) and 30 days for the three batches (Figure 9). Therefore, the analysis of variance (ANOVA) showed a statistically significant change (*p*-value = 0.00033; α = 0.05) for the period evaluated. This may be related to the small sediment (≈1 mm thick) observed in the visual analysis. However, in the later periods, Z-ave stability was observed, and between 30 and 90 days, there was no statistically significant difference (*p*-value = 0.94; α = 0.05). Therefore, its stability can be improved by increasing the viscosity of the formulation and/or adding components that increase the steric hindrance of the formulation, such as ammonium quaternary.

Additionally, a monomodal distribution was observed (Figure S6) after 90 days of incubation, with PdI ≤ 0.2, and no statistically significant change (*p*-value = 0.22239; α = 0.05) (Figure 9). Similarly, no significant changes were observed in pH (*p*-value = 0.78369; α = 0.05) and zeta potential (*p*-value = 0.18212; α = 0.05) (Figure 10). Additionally, the content was stable at the end of the period.

approximately 1 mm in thickness was seen at the bottom of the flasks; this sediment is

About Z-ave, an increase above 50 nm was observed between the initial analysis (time 0) and 30 days for the three batches (Figure 9). Therefore, the analysis of variance (ANOVA) showed a statistically significant change (*p*-value = 0.00033; α = 0.05) for the period evaluated. This may be related to the small sediment (≈1 mm thick) observed in the visual analysis. However, in the later periods, Z-ave stability was observed, and between 30 and 90 days, there was no statistically significant difference (*p*-value = 0.94; α

broken up easily through manual homogenization.

formulation, such as ammonium quaternary.

**Figure 9.** Determination of the average hydrodynamic diameter (Z-ave) in nm and polydispersion index (PdI), for the accelerated stability study of the nanosuspension of besifloxacin 0.6 wt%, in the time intervals: T0, 30, 60, and 90 days. Study condition: 40 °C ± 2 °C/75% RH ± 5% RH. *n* = 3 batch. **Figure 9.** Determination of the average hydrodynamic diameter (Z-ave) in nm and polydispersion index (PdI), for the accelerated stability study of the nanosuspension of besifloxacin 0.6 wt%, in the time intervals: T0, 30, 60, and 90 days. Study condition: 40 ◦C ± 2 ◦C/75% RH ± 5% RH. *n* = 3 batch. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 22 of 25

**Figure 10.** Determination of zeta potential (ZP) and pH for the accelerated stability study of the nanosuspension of besifloxacin 0.6 wt%, in the time intervals: T0, 30, 60, and 90 days. Study condition: 40 °C ± 2 °C/75% RH ± 5% RH. *n* = 3 batch. **Figure 10.** Determination of zeta potential (ZP) and pH for the accelerated stability study of the nanosuspension of besifloxacin 0.6 wt%, in the time intervals: T0, 30, 60, and 90 days. Study condition: 40 ◦C ± 2 ◦C/75% RH ± 5% RH. *n* = 3 batch.

#### **4. Conclusions 4. Conclusions**

The besifloxacin nanocrystals were successfully prepared using small-scale bed milling and response surface methodology. Povacoat® 0.9 wt% reduced the particle size of besifloxacin hydrochloride (550 nm) 17 times, which increased the saturation solubility by approximately two times concerning the raw material. The besifloxacin nanocrystal 0.6 wt% showed in vitro efficacy and did not show toxicity in a larval model. In addition, it presented satisfactory physical and chemical stability, and was able to meet the requirements of a commercial product. Thus, besifloxacin nanocrystals revealed the potential for reduced doses of the drug substance, with the minor occurrence of adverse effects and greater patient adherence to treatment. The besifloxacin nanocrystals were successfully prepared using small-scale bed milling and response surface methodology. Povacoat® 0.9 wt% reduced the particle size of besifloxacin hydrochloride (550 nm) 17 times, which increased the saturation solubility by approximately two times concerning the raw material. The besifloxacin nanocrystal 0.6 wt% showed in vitro efficacy and did not show toxicity in a larval model. In addition, it presented satisfactory physical and chemical stability, and was able to meet the requirements of a commercial product. Thus, besifloxacin nanocrystals revealed the potential for reduced doses of the drug substance, with the minor occurrence of adverse effects and greater patient adherence to treatment.

distribution by volume; (c) Correlogram; (d) Z-average trend; (e) Statistics graph *n* = 3; Figure S4: Xray diffraction of besifloxacin (raw material) and nanocrystals (freeze-dried). Figure S5: Toxicity of besifloxacin nanocrystals (500 mg/kg) and Povacoat® (750 mg/kg) in a larval model of *Galleria mellonella*; Figure S6: Particle distribution (monomodal), accelerated stability: 90 days. Study condition: 40°C ± 2°C/75% RH ± 5% RH; Table S1: Stability of the average hydrodynamic diameter (Z-ave) in nm, in the time intervals: immediately after milling (T0), 7 days, 15 days and 30 days. Process time 4 days. Storage condition: 25 °C. Table S2: Nano-based besifloxacin delivery: type,