Formulation and Evaluation of Nano Lipid Carrier-Based Ocular Gel System: Optimization to Antibacterial Activity

Sadaf Jamal Gilani; May Nasser bin Jumah; Ameeduzzafar Zafar; Syed Sarim Imam; Mohd Yasir; Mohammad Khalid; Sultan Alshehri; Mohammed M. Ghuneim; Fatima M. Albohairy

doi:10.3390/gels8050255

Gilani, Sadaf Jamal;Jumah, May Nasser bin;Zafar, Ameeduzzafar;Imam, Syed Sarim;Yasir, Mohd;Khalid, Mohammad;Alshehri, Sultan;Ghuneim, Mohammed M.;Albohairy, Fatima M.

2022-04-21 00:00:00

gels Article Formulation and Evaluation of Nano Lipid Carrier-Based Ocular Gel System: Optimization to Antibacterial Activity 1 2 , 3 , 4 5 , 6 , 7 Sadaf Jamal Gilani , May Nasser bin Jumah , Ameeduzzafar Zafar * , Syed Sarim Imam * , Mohd Yasir , 8 6 9 10 Mohammad Khalid , Sultan Alshehri , Mohammed M. Ghuneim and Fatima M. Albohairy Department of Basic Health Sciences, Preparatory Year, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia; sjglani@pnu.edu.sa Biology Department, College of Science, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia; mnbinjumah@pnu.edu.sa Environment and Biomaterial Unit, Health Sciences Research Center, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia Saudi Society for Applied Science, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, Jouf University, Sakaka 72341, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; salshehri1@ksu.edu.sa Department of Pharmacy, College of Health Science, Arsi University, Asella 396, Ethiopia; mohdyasir31@gmail.com Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia; drkhalid8811@gmail.com Department of Pharmacy Practice, College of Pharmacy, AlMaarefa University, Ad Diriyah 13713, Saudi Arabia; mghoneim@mcst.edu.sa Electron Microscope Research Unit, Health Sciences Research Center, Princes Nourah bint Abdulrahman Citation: Gilani, S.J.; Jumah, M.N.b.; University, Riyadh 11671, Saudi Arabia; fmalbohairy@pnu.edu.sa Zafar, A.; Imam, S.S.; Yasir, M.; * Correspondence: azafar@ju.edu.sa (A.Z.); simam@ksu.edu.sa (S.S.I.) Khalid, M.; Alshehri, S.; Ghuneim, M.M.; Albohairy, F.M. Formulation Abstract: The present research work was designed to prepare Azithromycin (AM)-loaded nano lipid and Evaluation of Nano Lipid carriers (NLs) for ocular delivery. NLs were prepared by the emulsiﬁcation–homogenization method Carrier-Based Ocular Gel System: and further optimized by the Box Behnken design. AM-NLs were optimized using the independent Optimization to Antibacterial constraints of homogenization speed (A), surfactant concentration (B), and lipid concentration (C) to Activity. Gels 2022, 8, 255. https:// obtain optimal NLs (AM-NLop). The selected AM-NLop was further converted into a sol-gel system doi.org/10.3390/gels8050255 using a mucoadhesive polymer blend of sodium alginate and hydroxyl propyl methyl cellulose (AM- Academic Editors: Maddalena NLopIG). The sol-gel system was further characterized for drug release, permeation, hydration, irrita- Sguizzato, Rita Cortesi and Rachel tion, histopathology, and antibacterial activity. The prepared NLs showed nano-metric size particles Yoon Chang (154.7 7.3 to 352.2 15.8 nm) with high encapsulation efﬁciency (48.8 1.1 to 80.9 2.9%). AM- Received: 1 March 2022 NLopIG showed a more prolonged drug release (98.6 4.6% in 24 h) than the eye drop (99.4 5.3% Accepted: 12 April 2022 in 3 h). The ex vivo permeation result depicted AM-NLopIG, AM-IG, and eye drop. AM-NLopIG Published: 21 April 2022 exhibited signiﬁcant higher AM permeation (60.7 4.1%) than AM-IG (33.46 3.04%) and eye drop Publisher’s Note: MDPI stays neutral (23.3 3.7%). The corneal hydration was found to be 76.45%, which is within the standard limit. The with regard to jurisdictional claims in histopathology and HET-CAM results revealed that the prepared formulation is safe for ocular use. published maps and institutional afﬁl- The antibacterial study revealed enhanced activity from the AM-NLopIG. iations. Keywords: NLs; in situ gel; ocular delivery; azithromycin; antibacterial; HET CAM Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article The treatment of ocular diseases is challenging due to the typical anatomy and phys- distributed under the terms and iology of the eye [1]. To attain the required amount of drug at the site of action at an conditions of the Creative Commons appropriate time is difﬁcult due to the dilution of formulations by tear ﬂuid, lachrymal Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ ﬂuid, eyelid blinking, tear ﬂuid turnover, and nasolacrimal drainage. Most ocular diseases 4.0/). are treated by eye drops, but only 5% of an administered dose is available for therapeutic Gels 2022, 8, 255. https://doi.org/10.3390/gels8050255 https://www.mdpi.com/journal/gels Gels 2022, 8, 255 2 of 18 action. The remaining dose was excreted out by various protection mechanisms. Therefore, the bioavailability of eye drops is low and they require multiple and frequent dosing, which may produce side effects [2,3]. The administration of topical non-invasive ocular formulations is the more preferable way to treat the anterior segment of the eye. To enhance the ocular bioavailability of the drugs, various novel drug carriers were investigated by the researchers. The delivery sys- tems include thymoquinone liposomes [4], pilocarpine niosomes [5], acetonide polymeric NPs [6], clarithromycin SLNs [7], methazolamide NLCs [8], besiﬂoxacin nanoemulsion [9], and tacrolimus in situ gel system [10]. Among these nano-delivery systems, NLCs have been widely used as potential car- riers belonging to lipid base nano-system [11]. NLCs are nano-metric size particles and give high entrapment efﬁciency due to lesser crystallinity of the lipid at low surfactant concentrations [12]. It also increases corneal permeability and bioavailability as well as reduces the local and systemic side effects. Various research studies have investigated NLCs for the treatment of ocular diseases [13,14]. Lakhani et al. formulated amphotericin B-loaded NLCs for ocular delivery and exhibited higher entrapment efﬁciency and higher antifungal activity than the marketed formulation [15]. In another study, Kiss et al. pre- pared dexamethasone NLCs for ocular inﬂammation. The prepared formulations exhibited a signiﬁcant drug concentration in the stroma layer, conﬁrmed by the porcine cornea study [16]. The application of NLCs was further explored by Seyfoddin and his research team and they developed acyclovir NLCs that exhibited higher entrapment efﬁciency than solid lipid nanoparticles as well as depicted high and faster permeation across the corneal membrane [17]. Besiﬂoxacin-loaded NLC formulations were evaluated for different parameters [18]. The prepared formulation showed nano-metric size, high entrapment efﬁciency, and signiﬁcant enhancement in corneal permeation. Due to the low viscosity of the NLC formulations, their efﬁcacy can be enhanced by transforming them into a sol-gel system. It is prepared in solution form and changes to gel phase in a cul-de-sac by temperature, ion, and pH [19]. Due to conversion into the gel phase, the increase in corneal residence time takes place. It leads to decreased dosing frequency and increases therapeutic efﬁcacy and may reduce the systemic side effects by minimizing the nasolacrimal outﬂow. Different research designs were reported for the NLC-based sol-gel system by the different administration routes. Ciproﬂoxacin-loaded NLC in situ gel was prepared and evaluated for the different parameters. The prepared formulations depicted 3.5-fold and 1.9-fold enhancements in the ﬂux and permeation compared to the marketed ciproﬂoxacin formulation [20]. In another study, moxiﬂoxacin NLC-laden in situ gel was developed and evaluated for endophthalmitis infection. The formulation exhibited a twofold higher permeation than pure drug solution [21]. Natamycin-loaded NLC in situ gel was prepared and optimized by the factorial design method. The formulations were prepared using guar gum, boric acid, and Carbopol 940 as a gelling agent. The prepared formulations depicted lower ﬂux value in comparison to Natacyn suspension [22]. Alginates are natural, nontoxic, mucoadhesive, viscosity enhancing polymers derived from different brown seaweeds [23]. They are used for ocular in situ gel systems due to their mucoadhesive, biocompatible, and non-toxic nature [24,25]. They have the properties of effective gelling capacity and viscosity and are commonly used in in situ gelling sys- tems [26]. The prepared ocular solution has a low viscosity at room temperature for easy administration and interacts with the calcium ions in the tear ﬂuid to form a gel matrix and lead to controlled drug release [27]. The alginates interact with divalent cations and bind to the guluronate blocks of the sodium alginate to facilitate gelation. The cross-linking of the cation–guluronate blocks then yields a gel-like structure, with a slow and steady drug release at the application site [28]. Draize ocular irritation studies on rabbits also reported sodium alginates to be nonirritating to the eye [23,29]. Hydroxypropyl methylcellulose (HPMC) is added into the formulation as a viscosity enhancer. It is a non-ionic, nontoxic, viscoelastic polymer with good swelling capacity [27,30]. Gels 2022, 8, x FOR PEER REVIEW 3 of 18 methylcellulose (HPMC) is added into the formulation as a viscosity enhancer. It is a non- ionic, nontoxic, viscoelastic polymer with good swelling capacity [27,30]. Azithromycin (AM) is a semisynthetic macrolide antibiotic. It is a broad-spectrum antibiotic and is highly stable in an acidic environment compared to other macrolides. It acts by inhibiting the 50s ribosomal subunit of bacteria and inhibits protein synthesis. It is commercially available on the market as eye drops. The present research work was designed with two steps. In the first step, AM-NLs were prepared by the emulsification homogenization method and further optimized by the Box Behnken design (BBD) using different independent variables. BBD-based opti- Gels 2022, 8, 255 3 of 18 mized AM-NLop was further converted into an in situ gel (sol-gel) system by using a thermosensitive gelling agent. The in situ gel was further characterized for viscosity, gel- Azithromycin (AM) is a semisynthetic macrolide antibiotic. It is a broad-spectrum ling capacity, in vitro release, ex vivo corneal permeation, ocular tolerance, and antimi- antibiotic and is highly stable in an acidic environment compared to other macrolides. It crobial evaluation. acts by inhibiting the 50s ribosomal subunit of bacteria and inhibits protein synthesis. It is commercially available on the market as eye drops. The present research work was designed with two steps. In the ﬁrst step, AM-NLs were 2. Result and Discussion prepared by the emulsiﬁcation homogenization method and further optimized by the Box Behnken design (BBD) using different independent variables. BBD-based optimized AM- 2.1. Screening of Solid and Liquid Lipids NLop was further converted into an in situ gel (sol-gel) system by using a thermosensitive gellingScreen agent. The ing o in f situ the sol gel was id further lipids, characterized liquid lipfor ids, viscosity and sur , gelling factants used capacity, for the development in vitro release, ex vivo corneal permeation, ocular tolerance, and antimicrobial evaluation. of NLs was performed based on the maximum solubility of AM, and the results are ex- 2. pressed Result and in F Discussion igure 1. The order of solubility of AM in various solid lipids is Glyceryl be- 2.1. Screening of Solid and Liquid Lipids henate > Stearic acid > Precirol ATO-5 > Tripalmitin > Glycerol monostearate > Myristic Screening of the solid lipids, liquid lipids, and surfactants used for the development acid > Glyceryl monooleate. The highest solubility of AM was found in GB (92.8 ± 6.2 of NLs was performed based on the maximum solubility of AM, and the results are ex- mg/g), and this was selected for further use as a solid lipid. In the case of liquid lipids, the pressed in Figure 1. The order of solubility of AM in various solid lipids is Glyceryl behenate > Stearic acid > Precirol ATO-5 > Tripalmitin > Glycerol monostearate > Myristic order of solubility of AM was found as follows: Miglyol > Labrasol > Sunflower oil > Ses- acid > Glyceryl monooleate. The highest solubility of AM was found in GB (92.8 6.2 mg/g), ame oil > Coconut oil > Isopropyl myristate. The maximum solubility of AM was found to and this was selected for further use as a solid lipid. In the case of liquid lipids, the order of solubility of AM was found as follows: Miglyol > Labrasol > Sunﬂower oil > Sesame oil be in Miglyol (76.3 ± 4.8 mg/mL), and this was selected as the optimal liquid lipid. The > Coconut oil > Isopropyl myristate. The maximum solubility of AM was found to be in order of solubility of AM in various surfactants is Kolliphor EL > Cremophor RH60 > Span Miglyol (76.3 4.8 mg/mL), and this was selected as the optimal liquid lipid. The order of 20 > Span 60. The highest solubility of AM was found to be in Kolliphor EL (83.1 ± 4.5 solubility of AM in various surfactants is Kolliphor EL > Cremophor RH60 > Span 20 > Span 60. The highest solubility of AM was found to be in Kolliphor EL (83.1 4.5 mg/mL). Based mg/mL). Based on the maximum solubility of AM, Glyceryl behenate, Miglyol, and Kolli- on the maximum solubility of AM, Glyceryl behenate, Miglyol, and Kolliphor EL were phor EL were used for the formulation of NLs. used for the formulation of NLs. Figure 1. Solubility proﬁle of Azithromycin in different lipids (solid and liquid) and surfactants. Figure 1. Solubility profile of Azithromycin in different lipids (solid and liquid) and surfactants. Study performed in triplicate and results shown as mean SD. Study performed in triplicate and results shown as mean ± SD. 2.2. Screening of Miscibility Ratio of Solid and Liquid Lipid The miscibility ratio (solid and liquid lipid) was determined by mixing the melted 2.2. Screening of Miscibility Ratio of Solid and Liquid Lipid solid and liquid lipid in different ratios, i.e., 9:1, 8:2, 7:3, 6:4, 5:5, and 2:8. Among the tested ratios, the combination 6:4 ratio was found to be the best blend because it did not show any The miscibility ratio (solid and liquid lipid) was determined by mixing the melted phase separation, oil droplets, or crystallization. So, this 6:4 ratio of solid and liquid lipids solid and liquid lipid in different ratios, i.e., 9:1, 8:2, 7:3, 6:4, 5:5, and 2:8. Among the tested was selected for the development of NLs. ratios, the combination 6:4 ratio was found to be the best blend because it did not show any phase separation, oil droplets, or crystallization. So, this 6:4 ratio of solid and liquid lipids was selected for the development of NLs. 2.3. Optimization Gels 2022, 8, 255 4 of 18 2.3. Optimization The Box Behnken design (BBD) depicted a total of seventeen compositions (ﬁve common) using the independent variables of homogenization speed (A), surfactant con- centration (B), and lipid concentration (C) at three different levels (low, medium, high), as shown in Table 1. The ﬁndings of the prepared AM-NLs were added to the software to obtain the optimized composition of independent constraints. Their effects were observed on the different models to obtain an optimized combination with desirable particle size and encapsulation efﬁciency. The impact of homogenization speed (A), surfactant concentration (B), and lipid concentration (C) was chosen based on a non-signiﬁcant lack of ﬁt and low predicted residual error sum of squares value (PRESS value) (Table 2). The polynomial equations were generated by ANOVA to explore the effect of homogenization speed (A), surfactant concentration (B), and lipid concentration (C) on responses (particle size and encapsulation efﬁciency). The positive and negative value of the polynomial equation indi- cates the synergistic and antagonistic effect on the dependent factors (responses). Among the different models of the responses, namely particle size (Y ) and entrapment efﬁciency (Y ), the quadratic model was found to be the best ﬁtting model [16]. Table 1. Composition of Azithromycin nano lipid carrier with experimental results. Independent Variables Dependent Variables Code Homogenization Entrapment Surfactant (%, w/v) Lipid (%, w/v) Particle Size (nm) Speed (rpm) Efﬁciency (%) A B C Y Y 1 2 AM-NL1 12,000 1.00 3.00 352.2 15.8 56.1 2.5 AM-NL2 20,000 1.00 3.00 228.5 09.2 54.3 2.1 AM-NL3 12,000 3.00 3.00 282.8 13.5 67.9 3.3 AM-NL4 20,000 3.00 3.00 210.3 08.7 63.3 1.9 AM-NL5 12,000 2.00 1.50 260.9 14.2 57.9 1.4 AM-NL6 20,000 2.00 1.50 165.6 6.8 55.7 1.8 AM-NL7 12,000 2.00 4.50 330.9 15.9 80.6 3.6 AM-NL8 20,000 2.00 4.50 220.0 11.2 76.8 2.9 AM-NL9 16,000 1.00 1.50 205.8 9.6 48.8 1.1 AM-NL10 16,000 3.00 1.50 154.7 07.3 56.9 2.1 AM-NL11 16,000 1.00 4.50 257.9 13.8 62.3 2.4 AM-NL12 16,000 3.00 4.50 222.2 11.7 80.9 2.9 * AM-NL13 16,000 2.00 3.00 176.1 12.4 75.4 2.6 * AM-NL14 16,000 2.00 3.00 176.1 12.4 73.4 2.4 * AM-NL15 16,000 2.00 3.00 180.5 11.9 75.4 2.6 * AM-NL16 16,000 2.00 3.00 176.1 12.4 73.4 2.4 * AM-NL17 16,000 2.00 3.00 180.5 11.9 75.4 2.6 * Center point. 2.4. Inﬂuence of Independent Variables on Particle Size (Y ) The mean particle size of the prepared AM-NLs was between 154.7 7.3 (AM-NL10) and 352.2 15.8 nm (AM-NL1), as shown in Table 1. The minimum particle size (Y ) was found from the formulation (AM-NL10) prepared with homogenization speed of 16,000 rpm, surfactant concentration of 3% w/v, and lipid concentration of 1.5% w/v. The maximum particle size (Y ) was found from the formulation (AM-NL1) prepared with composition homogenization speed of 12,000 rpm, surfactant concentration of 1% w/v, and lipid concentration of 3% w/v. From the results, there is a signiﬁcant difference in the particle size was observed by changing the composition of independent variables. The effects of independent variables (homogenization speed, surfactant concentration, and lipid concentration) were evaluated on the 3D response surface plot (Figure 2). As shown in Figure 2, homogenization speed (A) showed a dual effect on particle size. The increase in the homogenization speed from a low level to a high level caused the particle size to decrease. After reaching an intermediate level, the further increase in the homogenization Gels 2022, 8, x FOR PEER REVIEW 5 of 18 R² 0.6458 0.6640 0.9918 Adjusted R² 0.5640 0.4623 0.9812 Predicted R² 0.4364 0.0131 0.9013 Entrapment ef- %CV - - 2.14 Gels 2022, 8, 255 5 of 18 ficiency Adeq. Precision −983.49 1722.20 172.20 68.79 97.85 3.49 F-value Significant Significant Non-Significant speeds results in increased particle size. The increase in size may take place due to the p-value 0.0005 0.0003 0.1295 aggregation of particles at a higher homogenization speed. The small globules of emulsion aggregate in the inter-particular space of big globules, leading to the re-coalescence or 2.4. Influence of Independent Variables on Particle Size (Y1) Ostwald ripening. Besides this, surfactant (B) was responsible for the decrease in interfacial tension, and as per the Laplaces pressure theory, as the interfacial tension decrease, more The mean particle size of the prepared AM-NLs was between 154.7 ± 7.3 (AM-NL10) droplets are disrupted into ﬁne particles. Hence, the surfactant concentration exhibited a and 352.2 ± 15.8 nm (AM-NL1), as shown in Table 1. The minimum particle size (Y1) was negative effect on particle size. Similar results are reported in the literature [21]. At a ﬁxed found from the formulation (AM-NL10) prepared with homogenization speed of 16,000 homogenization speed (A) and surfactant concentration (B), the particle size (Y ) increases rpm, surfactant concentration of 3% w/v, and lipid concentration of 1.5% w/v. The maxi- with increased lipid concentration. This might be due to an enhancement in the viscosity of mum particle size (Y1) was found from the formulation (AM-NL1) prepared with compo- lipid dispersion [31]. Moreover, there was a high chance of incomplete emulsiﬁcation at a high lipid concentration and low surfactant concentration (due to lack of surfactant), which sition homogenization speed of 12,000 rpm, surfactant concentration of 1% w/v, and lipid was responsible for the aggregation of lipid nanoparticles. The controlled optimal particle concentration of 3% w/v. From the results, there is a significant difference in the particle size is good for the stability of nano-dispersion as it promotes the Brownian motion and size was observed by changing the composition of independent variables. The effects of prevents sedimentation [18]. independent variables (homogenization speed, surfactant concentration, and lipid con- Table 2. Statistical model ﬁt summary report. centration) were evaluated on the 3D response surface plot (Figure 2). As shown in Figure 2, homogenization speed (A) showed a dual effect on particle size. The increase in the Parameters Regression Parameters Models Model homogenization speed from a low level to a high level caused the particle size to decrease. Linear 2FI Quadratic After reaching an intermediate level, the further increase in the homogenization speeds SD 40.76 45.64 2.71 results in increased particle size. The increase in size may take place due to the aggrega- R 0.5929 0.6074 0.9990 tion of particles at a higher homogenization speed. The 0.4990 small g 0.3718 lobules of emulsio 0.9978 n aggre- Adjusted R Particle size Predicted R 0.3270 0.2725 0.9941 Quadratic gate in the inter-particular space of big globules, leading to the re-coalescence or Ostwald %CV - - 1.22 ripening. Besides this, surfactant (B) was responsible for the decrease in interfacial tension, Ade Precision 35,703.02 67,503.77 313.33 and as per the Laplaces pressure theory, as the interfacial tension decrease, more droplets 272.57 394.27 0.61 F-value Signiﬁcant Signiﬁcant Non-Signiﬁcant Lack of ﬁt are disrupted into fine particles. Hence, the surfactant concentration exhibited a negative p-value <0.0001 <0.0001 0.6419 effect on particle size. Similar results are reported in the literature [21]. At a fixed homog- SD 6.90 7.66 1.43 enization speed (A) and surfactant concentration (B), the particle size (Y1) increases with R 0.6458 0.6640 0.9918 increased lipid concentration. This might be due to an enhancement in the viscosity of Adjusted R 0.5640 0.4623 0.9812 Predicted R 0.4364 0.0131 0.9013 lipid dispersion [31]. Moreover, there was a high chance of incomplete emulsification at a Entrapment efﬁciency Quadratic %CV - - 2.14 high lipid concentration and low surfactant concentration (due to lack of surfactant), Adeq. Precision 983.49 1722.20 172.20 which was responsible for the aggregation of lipid nanoparticles. The controlled optimal 68.79 97.85 3.49 F-value Signiﬁcant Signiﬁcant Non-Signiﬁcant Lack of ﬁt particle size is good for the stability of nano-dispersion as it promotes the Brownian mo- p-value 0.0005 0.0003 0.1295 tion and prevents sedimentation [18]. Figure 2. Effect of independent variables (Homogenization speed—A, Surfactant—B, Lipid—C) on Figure 2. Effect of independent variables (Homogenization speed—A, Surfactant—B, Lipid—C) on the particle size (Y1). the particle size (Y ). Lack of fit Gels 2022, 8, 255 6 of 18 The effect of independent variables was further statistically analyzed by the polyno- mial Equation (1). 2 2 2 Particle size (Y ) = +178.2 50.27A 21.8B + 30.48C + 12.75AB 3.90AC + 3.80BC + 62.24A + 27.99B + 3.94C (1) The above equation states that homogenization speed (A, coefﬁcient 50.27) and surfactant (B, coefﬁcient 21.80) showed a negative effect. On the other hand, the third variable, lipid concentration (C, coefﬁcient +30.48), depicted a positive effect. The quadratic 2 2 2 coefﬁcient of A , B , and C showed the positive values of +62.24, 27.99, and +3.94, re- spectively. The interpretation explains that the variables alone and in combination had a positive effect on the particle size. Based on insigniﬁcant lack of ﬁt (F = 0.61, P = 0.6419) and the lowest precision (313.33) value, the quadratic model was chosen as the best ﬁtting model to describe the inﬂuence of independent variables on particle size. 2.5. Inﬂuence of Independent Variables on Entrapment Efﬁciency (Y ) The mean entrapment efficiency of the prepared AM-NLs was found between 48.8 1.1 (AM-NL9) and 80.9 2.9 (AM-NL12), as shown in Table 1. The minimum entrapment efﬁciency (Y ) was found from the formulation (AM-NL9) prepared with homogenization speed of 16,000 rpm, surfactant concentration of 1% w/v, and lipid concentration of 1.5% w/v. The maximum particle size (Y ) was found from the formulation (AM-NL12) prepared with composition homogenization speed of 16,000 rpm, surfactant concentration of 3% w/v, and lipid concentration of 4.5% w/v. From the results, a signiﬁcant difference in the entrapment efﬁciency was observed by changing the composition of independent variables. The effects of independent variables (homogenization speed, surfactant concentration, and lipid con- centration) were evaluated on the 3D response surface plot (Figure 3). As shown in Figure 3, homogenization speed (A) showed a synergistic effect on entrapment efﬁciency. With the increase in the homogenization speed from a low level (12,000 rpm) to an intermediate level (16,000 rpm), AM entrapment increased. After reaching the intermediate level, the further increase in the homogenization speed decreased the entrapment efﬁciency. The decrease in entrapment efﬁciency may be due to the leaching of drugs at a high homogenization speed. The surfactant (B) also showed a dual effect on the entrapment efﬁciency. The increase in surfactant concentration led to a decrease in interfacial tension and a greater amount of solubilized and entrapped AM. The presence of a sufﬁcient surfactant concentration pre- vents the drug expulsion and ultimately forms stable nanoparticles. Contrarily, if sufﬁcient lipids are not available for drug entrapment, the reverse results will occur [32]. The third variable, lipid concentration (C), depicted a positive effect on entrapment efﬁciency. The increase in lipid content resulted in a greater amount of AM entrapped inside the NLs. The possible reason for this is the availability of more space for the accommodation of drugs within the NL vesicles [33]. A low concentration of surfactants and high concentration of lipids gives lesser entrapment due to incomplete emulsiﬁcation. The effect of independent variables was further evaluated by the polynomial equation given below: 2 2 2 Entrapment efficiency = +74.71 1.62A + 5.98B + 10.13C + 0.70AB 0.40AC + 2.70BC 4.42A 10.02B 2.51C (2) As depicted in Equation (2), homogenization speed with the magnitudes –1.62 (A) and 4.42 (A ) exhibited a negative effect on entrapment efﬁciency. On the other hand, upon increasing the surfactant concentration (magnitude +5.98), the entrapment efﬁciency was increased if sufﬁcient lipids were present in the dispersion. This might be due to the solubility of the drug in the presence of sufﬁcient surfactants. The quadratic model was considered as the best ﬁtting model based on non-signiﬁcant lack of ﬁt (F = 3.49, p-value 0.1295) and lowest precision (172.2) value (Table 2). It described the inﬂuence of independent variables on entrapment efﬁciency. Gels 2022 Gels ,2022 8, x FO , 8, 255 R PEER REVIEW 7 of 18 7 of 18 Figure 3. Effects of independent variables (Homogenization speed - A, Surfactant - B, Lipid - C) on Figure 3. Effects of independent variables (Homogenization speed—A, Surfactant—B, Lipid—C) on the encapsulation efficiency (Y2). the encapsulation efﬁciency (Y ). 2.6. Optimized Composition The effect of independent variables was further evaluated by the polynomial equa- tion given below: Table 2 and Figures 2 and 3 indicate that all the process and formulation variables were well controlled, and the obtained values were in an acceptable 2range. Based 2 on the 2 above Entrapment efficiency = +74.71 − 1.62A + 5.98B + 10.13C + 0.70AB − 0.40AC + 2.70BC − 4.42A − 10.02B − 2.51C (2) results, it was concluded that all three variables had a signiﬁcant effect on the responses. As depicted in Equation (2), homogenization speed with the magnitudes –1.62 (A) Among the 17 prepared formulations, AM-NL13 was selected for further point prediction and −4.42 (A ) exhibited a negative effect on entrapment efficiency. On the other hand, optimization by slightly changing the composition and further evaluated for particle size upon increasing the surfactant concentration (magnitude +5.98), the entrapment efficiency and entrapment efﬁciency (Table 3). The practical results of particle size and entrapment was increased if sufficient lipids were present in the dispersion. This might be due to the efﬁciency were found to be closer to each other, and the overall desirability was found to solubility of the drug in the presence of sufficient surfactants. The quadratic model was be 0.992, which conﬁrms the validity of the model. considered as the best fitting model based on non-significant lack of fit (F = 3.49, P-value 0.1295) and lowest precision (172.2) value (Table 2). It described the influence of inde- Table 3. Point prediction optimization by Box Behnken design. pendent variables on entrapment efficiency. Actual Value Predicted Value Homogenization Speed: Surfactant: Lipid Code 2.6. Optimized Composition Y (nm) Y (%) Y (nm) Y (%) 1 2 1 2 Table 2 and Figures 2 and 3 indicate that all the process and formulation variables AM-NL13 16,000:2:3 176.1 12.4 75.4 2.6 178.20 74.7 AM-NLop were well co 16,500:2.25:3 ntrolled, and the obtained 154.1values 6.35 were 78.15 in an accep 1.9 table ran 161.3ge. Based on t 79.8he above results, it was concluded that all three variables had a significant effect on the re- sponses. Among the 17 prepared formulations, AM-NL13 was selected for further point The selected optimized formulation (AM-NLop) was the composition prepared with prediction optimization by slightly changing the composition and further evaluated for homogenization speed of 16,500 rpm, surfactant concentration of 2.25% w/v, and lipid con- particle size and entrapment efficiency (Table 3). The practical results of particle size and centration of 3% w/v. It showed particle size and entrapment efﬁciency of 154.1 6.35 nm entrapment efficiency were found to be closer to each other, and the overall desirability (Figure 4) and 78.15 1.9%. It depicted a PDI value of 0.18 and negative zeta potential was found to be 0.992, which confirms the validity of the model. (34.24 mV), indicating higher stability. The low PDI (less than 0.5) and high negative zeta potential (30 mV) revealed the greater homogeneity of particles. The predicted value of Table 3. Point prediction optimization by Box Behnken design. Gels 2022, 8, x FOR PEER REVIEW 8 of 18 particle size and entrapment efﬁciency was found to be 161.3 nm and 79.8%, and the value was found to be closer to the predicted values. Homogenization Speed: Actual Value Predicted Value Code Surfactant: Lipid Y1 (nm) Y2 (%) Y1 (nm) Y2 (%) AM-NL13 16,000:2:3 176.1 ± 12.4 75.4 ± 2.6 178.20 74.7 AM-NLop 16,500:2.25:3 154.1 ± 6.35 78.15 ± 1.9 161.3 79.8 The selected optimized formulation (AM-NLop) was the composition prepared with homogenization speed of 16,500 rpm, surfactant concentration of 2.25% w/v, and lipid concentration of 3% w/v. It showed particle size and entrapment efficiency of 154.1 ± 6.35 nm (Figure 4) and 78.15 ± 1.9%. It depicted a PDI value of 0.18 and negative zeta potential (−34.24 mV), indicating higher stability. The low PDI (less than 0.5) and high negative zeta potential (±30 mV) revealed the greater homogeneity of particles. The predicted value of particle size and entrapment efficiency was found to be 161.3 nm and 79.8%, and the value was found to be closer to the predicted values. Figure 4. Particle size distribution image of Azithromycin-loaded nano lipid carrier (AM-NLop). Figure 4. Particle size distribution image of Azithromycin-loaded nano lipid carrier (AM-NLop). 2.7. X-ray Diffraction (XRD) Analysis Figure 5 shows the diffractogram of AM and AM-NLop. AM exhibited a characteris- tic highly intense peak at 2-theta levels of 8.2°, 10.2°, 13°, 16.2°, 17.2°, 19.1°, and 29.6°, assuring its crystallinity. The diffractogram of AM-NLop did not exhibit any characteristic sharp peak of AM after encapsulating into NLs. The low intensity and broad AM peaks indicate that AM was encapsulated or solubilized in a lipid matrix. The reduction in the intensities takes place due to some modification in the crystallinity in the NLs, attributed to the disordering of the solid lipid crystalline structure due to the presence of liquid lipids [34]. Figure 5. XRD image of Azithromycin and Azithromycin-loaded nano lipid carrier (AM-NLop). 2.8. Gel Evaluation 2.8.1. Clarity and Gelling Ability The formulation was found to be clear upon observation with a black and white back- ground. The presence of foreign particles may produce irritation to the soft tissue of the ocular region. It was also evaluated for sol to gel properties after gradually converting into a gel after the addition of the sol form into STF. This gelling is due to the replacement of sodium from sodium alginate with Ca in tear fluid, forming calcium alginate. Table 4 shows the viscosity results of AM-NLopG and AM-G formulations. The viscosity of AM- NLopG in the solution state was found to be very low, whereas after conversion into gel form, a significant enhancement in the viscosity was observed. The viscosity was also evaluated for the control formulation AM-IG. In solution form, it shows the viscosity of 235.34 ± 7.21 cps, and after conversion into gel state, it showed 495.18 ± 8.72 cps. AM- Gels 2022, 8, x FOR PEER REVIEW 8 of 18 Figure 4. Particle size distribution image of Azithromycin-loaded nano lipid carrier (AM-NLop). 2.7. X-ray Diffraction (XRD) Analysis Gels 2022, 8, 255 8 of 18 Figure 5 shows the diffractogram of AM and AM-NLop. AM exhibited a characteris- tic highly intense peak at 2-theta levels of 8.2°, 10.2°, 13°, 16.2°, 17.2°, 19.1°, and 29.6°, 2.7. X-ray Diffraction (XRD) Analysis assuring its crystallinity. The diffractogram of AM-NLop did not exhibit any characteristic Figure 5 shows the diffractogram of AM and AM-NLop. AM exhibited a characteristic sharp peak of AM after encapsulating into NLs. The low intensity and broad AM peaks highly intense peak at 2-theta levels of 8.2 , 10.2 , 13 , 16.2 , 17.2 , 19.1 , and 29.6 , assuring indicate that AM was encapsulated or solubilized in a lipid matrix. The reduction in the its crystallinity. The diffractogram of AM-NLop did not exhibit any characteristic sharp intensities takes place due to some modification in the crystallinity in the NLs, attributed peak of AM after encapsulating into NLs. The low intensity and broad AM peaks indicate that AM was encapsulated or solubilized in a lipid matrix. The reduction in the intensities to the disordering of the solid lipid crystalline structure due to the presence of liquid lipids takes place due to some modiﬁcation in the crystallinity in the NLs, attributed to the [34]. disordering of the solid lipid crystalline structure due to the presence of liquid lipids [34]. Figure 5. XRD image of Azithromycin and Azithromycin-loaded nano lipid carrier (AM-NLop). Figure 5. XRD image of Azithromycin and Azithromycin-loaded nano lipid carrier (AM-NLop). 2.8. Gel Evaluation 2.8.1. 2.8. Gel Clarity Ev and aluGelling ation Ability The formulation was found to be clear upon observation with a black and white 2.8.1. Clarity and Gelling Ability background. The presence of foreign particles may produce irritation to the soft tissue of the ocular region. It was also evaluated for sol to gel properties after gradually converting The formulation was found to be clear upon observation with a black and white back- into a gel after the addition of the sol form into STF. This gelling is due to the replacement ground. The presence of foreign particles may produce irritation to the soft tissue of the of sodium from sodium alginate with Ca in tear ﬂuid, forming calcium alginate. Table 4 ocular region. It was also evaluated for sol to gel properties after gradually converting shows the viscosity results of AM-NLopG and AM-G formulations. The viscosity of AM- NLopG in the solution state was found to be very low, whereas after conversion into gel into a gel after the addition of the sol form into STF. This gelling is due to the replacement form, a signiﬁcant enhancement in the viscosity was observed. The viscosity was also of sodium from sodium alginate with Ca in tear fluid, forming calcium alginate. Table 4 evaluated for the control formulation AM-IG. In solution form, it shows the viscosity shows the viscosity results of AM-NLopG and AM-G formulations. The viscosity of AM- of 235.34 7.21 cps, and after conversion into gel state, it showed 495.18 8.72 cps. AM-NLopG showed higher viscosity in the solution state due to the presence of different NLopG in the solution state was found to be very low, whereas after conversion into gel ingredients used to prepare NLs, and after converting into gel state, greater viscosity was form, a significant enhancement in the viscosity was observed. The viscosity was also also observed due to higher gelling capacity. evaluated for the control formulation AM-IG. In solution form, it shows the viscosity of Table 4. Physicochemical characterization of in situ gel. 235.34 ± 7.21 cps, and after conversion into gel state, it showed 495.18 ± 8.72 cps. AM- AM-G AM-NLopG Physicochemical Parameters Sol State Gel State Sol State Gel State Viscosity (cps) 131.25 6.23 402.12 5.52 235.34 7.2 495.18 8.7 Cohesiveness (g) 0.8 0.02 0.94 0.01 0.78 0.09 0.86 0.05 Adhesiveness (N-mm) 13.34 0.92 26.23 1.52 14.24 1.1 29.43 1.1 Hardness (N) 3.54 0.32 8.34 0.38 3.21 0.3 8.76 0.5 Gels 2022, 8, x FOR PEER REVIEW 9 of 18 NLopG showed higher viscosity in the solution state due to the presence of different in- gredients used to prepare NLs, and after converting into gel state, greater viscosity was also observed due to higher gelling capacity. Table 4. Physicochemical characterization of in situ gel. AM-G AM-NLopG Physicochemical Parameters Sol State Gel State Sol State Gel State Viscosity (cps) 131.25 ± 6.23 402.12 ± 5.52 235.34 ± 7.2 495.18 ± 8.7 Cohesiveness (g) 0.8 ± 0.02 0.94 ± 0.01 0.78 ± 0.09 0.86 ± 0.05 Adhesiveness (N-mm) 13.34 ± 0.92 26.23 ± 1.52 14.24 ± 1.1 29.43 ± 1.1 Hardness (N) 3.54 ± 0.32 8.34 ± 0.38 3.21 ± 0.3 8.76 ± 0.5 2.8.2. Texture Analysis Texture analysis of AM-NLopIG and AM-IG (control) formulation was performed to determine the mechanical properties, and the results are expressed in Table 4. The data show that cohesiveness, adhesiveness, and hardness of AM-NLopIG were found to be significantly changed (p < 0.05) in sol and gel states. It showed cohesiveness of 0.78 ± 0.12 Gels 2022, 8, 255 9 of 18 (sol) to 0.86 ± 0.09 (gel), adhesiveness of 14.24 ± 1.03 (sol) to 29.43 ± 1.12 (gel), and hardness of 3.21 ± 0.24 (sol) to 8.76 ± 0.51 (gel). The results of the mechanical properties of AM- 2.8.2. NLop Textur G and e Analysis AM-IG did not show significant changes (p > 0.05). Interaction of Ca with Texture analysis of AM-NLopIG and AM-IG (control) formulation was performed the gelling polymer in situ gel system directly affects mechanical properties. The higher to determine the mechanical properties, and the results are expressed in Table 4. The value of adhesiveness indicates more adhesion to the corneal surface and increases the data show that cohesiveness, adhesiveness, and hardness of AM-NLopIG were found residence time. However, the cohesiveness of the formulation reduces the irritation and to be signiﬁcantly changed (p < 0.05) in sol and gel states. It showed cohesiveness of 0.78 0.12 (sol) to 0.86 0.09 (gel), adhesiveness of 14.24 1.03 (sol) to 29.43 1.12 (gel), makes it easy to spread on the ocular surface [35]. and hardness of 3.21 0.24 (sol) to 8.76 0.51 (gel). The results of the mechanical properties of AM-NLopG and AM-IG did not show signiﬁcant changes (p > 0.05). Interaction of Ca 2.8.3. In Vitro Drug Release with the gelling polymer in situ gel system directly affects mechanical properties. The higher value of adhesiveness indicates more adhesion to the corneal surface and increases Figure 6 depicts the comparative release profile of AM-NLopIG, AM-IG, and eye the residence time. However, the cohesiveness of the formulation reduces the irritation and drop using the dialysis bag method. The release of AM from AM-NLopIG and AM-IG makes it easy to spread on the ocular surface [35]. was found to be 98.65 ± 4.65% and 49.48 ± 4.23%, respectively, in 24 h. The eye drop de- 2.8.3. In Vitro Drug Release picted 99.45 ± 5.3% release in 3 h. AM-NLopIG exhibited biphasic release behavior, with Figure 6 depicts the comparative release proﬁle of AM-NLopIG, AM-IG, and eye drop using initia the l fa dialysis st relea bag se a method. nd laThe ter sl release ow rel of AM ease. from Th AM-NLopIG e initial fast re and AM-IG lease was may be due to the nano- found to be 98.65 4.65% and 49.48 4.23%, respectively, in 24 h. The eye drop depicted sized particles of NLs and a higher effective surface area. The drug particles adsorbed on 99.45 5.3% release in 3 h. AM-NLopIG exhibited biphasic release behavior, with initial the surface of NLs enter the dissolution media. Later, the slower drug release was found fast release and later slow release. The initial fast release may be due to the nano-sized particles of NLs and a higher effective surface area. The drug particles adsorbed on the to be due to the formation of gel matrix, and the drug needs to cross it as well as the surface of NLs enter the dissolution media. Later, the slower drug release was found to dialysis membrane. The eye drop showed quicker release in 3 h due to the lesser viscosity, be due to the formation of gel matrix, and the drug needs to cross it as well as the dialysis and the drug is available for release. AM-IG showed significantly less release than AM- membrane. The eye drop showed quicker release in 3 h due to the lesser viscosity, and the drug is available for release. AM-IG showed signiﬁcantly less release than AM-NLopIG due NLopIG due to the poor solubility and non-availability of surfactants. The surfactant to the poor solubility and non-availability of surfactants. The surfactant helps to increase helps to increase the solubility and lowers the interfacial tension between the two phases. the solubility and lowers the interfacial tension between the two phases. Figure 6. In vitro release data of the Azithromycin nano lipid carrier (AM-NLop), Azithromycin nano lipid carrier-based in situ gel (AM-NLopIG), and AM eye drop. The tests were performed in triplicate and data are shown as mean SD. 2.8.4. Ex Vivo Permeation Study The comparative ex vivo permeation results of AM-NLopIG, AM-IG, and eye drop showed signiﬁcant differences in permeation (%) and ﬂux. AM-NLopIG exhibited signiﬁ- cantly (p < 0.05) higher permeation (60.76 4.12%) than AM-IG (33.46 3.04%) and eye drop (23.31 3.76%). The ﬂux of AM-NLopIG was found to be 153.43 g/h.cm , which is 1.82-fold higher than AM-IG and 2.6-fold higher than the eye drop. The higher permeation Gels 2022, 8, x FOR PEER REVIEW 10 of 18 Figure 6. In vitro release data of the Azithromycin nano lipid carrier (AM-NLop), Azithromycin nano lipid carrier-based in situ gel (AM-NLopIG), and AM eye drop. The tests were performed in triplicate and data are shown as mean ± SD. 2.8.4. Ex Vivo Permeation Study The comparative ex vivo permeation results of AM-NLopIG, AM-IG, and eye drop showed significant differences in permeation (%) and flux. AM-NLopIG exhibited signif- icantly (p < 0.05) higher permeation (60.76 ± 4.12%) than AM-IG (33.46 ± 3.04%) and eye drop (23.31 ± 3.76%). The flux of AM-NLopIG was found to be 153.43 µg/h.cm , which is 1.82-fold higher than AM-IG and 2.6-fold higher than the eye drop. The higher permeation of AM from AM-NLopIG was found due to the increase in AM solubility after nano-siz- ing. The presence of lipids and surfactants of NLs increases the endocytosis through the corneal epithelium and leads to greater permeation across the membrane [21,36]. The sol form of a prepared formulation is converted to gel form and the contact time increases. The increase in contact time and the used polymer helps to open the tight junction of the Gels 2022, 8, 255 membrane and gives greater permeation. The apparent permeabi 10 oflit 18y coefficients of AM- −1 −1 NLopIG, AM-IG, and eye drop were calculated as 2.4 × 10 cm/sec, 1.3 × 10 cm/sec, and −2 9.3 × 10 cm/sec, respectively. AM-NLopIG showed an enhancement ratio 1.82-fold higher of AM from AM-NLopIG was found due to the increase in AM solubility after nano-sizing. than AM-IG and 2.61-fold higher than the eye drop. The presence of lipids and surfactants of NLs increases the endocytosis through the corneal epithelium and leads to greater permeation across the membrane [21,36]. The sol form of a prepared formulation is converted to gel form and the contact time increases. The increase 2.8.5. Corneal Hydration in contact time and the used polymer helps to open the tight junction of the membrane and The ocular hydration study was performed to evaluate the ocular toxicity after keep- gives greater permeation. The apparent permeability coefﬁcients of AM-NLopIG, AM-IG, 1 1 2 and eye drop were calculated as 2.4 10 cm/s, 1.3 10 cm/s, and 9.3 10 cm/s, ing the formulation with the cornea. After treatment, the corneal hydration was found to respectively. AM-NLopIG showed an enhancement ratio 1.82-fold higher than AM-IG and be 76.45%, which is within the standard limit of 76−80% [37]. The results of the study re- 2.61-fold higher than the eye drop. vealed that AM-NLopIG did not produce any toxicity and did not alter the structure of 2.8.5. Corneal Hydration the cornea. There was no damage to the cornea observed externally and internally, which The ocular hydration study was performed to evaluate the ocular toxicity after keeping was further confirmed by histopathology examination. the formulation with the cornea. After treatment, the corneal hydration was found to be 76.45%, which is within the standard limit of 76–80% [37]. The results of the study revealed that AM-NLopIG did not produce any toxicity and did not alter the structure of the cornea. 2.8.6. Histopathology There was no damage to the cornea observed externally and internally, which was further The cornea was collected after the permeation study and assessed for internal dam- conﬁrmed by histopathology examination. age in the structure. The histopathology of the treated cornea with AM-NLopIG and con- 2.8.6. Histopathology trol (NaCl, 0.9%) was compared to changes in the structure, as depicted in Figure 7A,B. The cornea was collected after the permeation study and assessed for internal damage AM-NLopIG-treated cornea revealed no marked change in the internal structure. No in the structure. The histopathology of the treated cornea with AM-NLopIG and control damage in cellular structure or histological alterations were observed in the treated cor- (NaCl, 0.9%) was compared to changes in the structure, as depicted in Figure 7A,B. AM- NLopIG-treated cornea revealed no marked change in the internal structure. No damage in nea, matching with the normal saline-treated cornea. The epithelium, Bowman’s mem- cellular structure or histological alterations were observed in the treated cornea, matching brane, and stroma of the cornea and the cells were found intact, similar to the control with the normal saline-treated cornea. The epithelium, Bowman’s membrane, and stroma sample. The morphology of the cornea was also well maintained [38]. From the results, of the cornea and the cells were found intact, similar to the control sample. The morphology of the cornea was also well maintained [38]. From the results, we can presume that there we can presume that there is no alteration in the corneal structure, and that AM-NLopIG is no alteration in the corneal structure, and that AM-NLopIG is nontoxic and compatible is nontoxic and compatible with the ocular structure. with the ocular structure. Figure 7. Histopathology image of AM-NLopIG-treated cornea (A). NaCl-treated cornea (B) taken by Figure 7. Histopathology image of AM-NLopIG-treated cornea (A). NaCl-treated cornea (B) taken light microscope at image scale 40. by light microscope at image scale 40×. 2.8.7. HET-CAM Irritation Study HET-CAM (hen’s egg chorioallantoic membrane) is an in vitro evaluation method. It is helps to determine the irritation potential of formulations and also used as an alternative to the Draize test in rabbits. The results of CAM treated with AM-NLopIG, a negative control (0.9% NaCl), and a positive control (1% SLS) are expressed in Figure 8A–C. AM-NLopIG and the negative control did not exhibit any damage to CAM (blood capillaries). The score Gels 2022, 8, x FOR PEER REVIEW 11 of 18 2.8.7. HET-CAM Irritation Study HET-CAM (hen’s egg chorioallantoic membrane) is an in vitro evaluation method. It is helps to determine the irritation potential of formulations and also used as an alterna- tive to the Draize test in rabbits. The results of CAM treated with AM-NLopIG, a negative control (0.9% NaCl), and a positive control (1% SLS) are expressed in Figure 8A–C. AM- Gels 2022, 8, x FOR PEER REVIEW 11 of 18 NLopIG and the negative control did not exhibit any damage to CAM (blood capillaries). The score was found to be closer to zero and considered as non-irritant. It revealed no damage to the vein or arter 2.8.7. HET-CAM Irritation Study y, no bleeding and hemorrhage observed. The positive control depicted HET-CAM a hi(gh ir hen’s egg chorioa ritant cumulative llantoic membra scor ne) is e of an 22.33 in vitr(se o evalua vere irritant tion method. It ) with bleed ing and ex- is helps to determine the irritation potential of formulations and also used as an alterna- cessive hemorrhage. From the results, it can be concluded that the prepared formulation Gels 2022, 8, 255 11 of 18 tive to the Draize test in rabbits. The results of CAM treated with AM-NLopIG, a negative AM-NLopIG is safe and non-irritant for ocular administration. control (0.9% NaCl), and a positive control (1% SLS) are expressed in Figure 8A–C. AM- NLopIG and the negative control did not exhibit any damage to CAM (blood capillaries). was found to be closer to zero and considered as non-irritant. It revealed no damage to the The score was found to be closer to zero and considered as non-irritant. It revealed no vein or artery, no bleeding and hemorrhage observed. The positive control depicted a high damage to the vein or artery, no bleeding and hemorrhage observed. The positive control irritant cumulative score of 22.33 (severe irritant) with bleeding and excessive hemorrhage. depicted a high irritant cumulative score of 22.33 (severe irritant) with bleeding and ex- From the results, it can be concluded that the prepared formulation AM-NLopIG is safe cessive hemorrhage. From the results, it can be concluded that the prepared formulation and non-irritant for ocular administration. AM-NLopIG is safe and non-irritant for ocular administration. Figure 8. HET-CAM irritation test image treated with AM-NLopIG (A), 0.9% NaCl (B), and 1% SLS (C). Figure 8. HET-CAM irritation test image treated with AM-NLopIG (A), 0.9% NaCl (B), and 1% SLS (C). Figure 8. HET-CAM irritation test image treated with AM-NLopIG (A), 0.9% NaCl (B), and 1% 2.8.8. Isotonicity Study SLS (C). 2.8.8. Isotonicity Study The study was performed to evaluate the damage to the blood cells after treatment 2.8.8. Isotonicity Study The study was performed to evaluate the damage to the blood cells after treatment with AM-NLopIG. It was evaluated by treating with goat blood and observed under a with AM-NLopIG. It was evaluated by treating with goat blood and observed under a The study was performed to evaluate the damage to the blood cells after treatment microscope for any damage to RBCs. AM-NLopIG- and control (0.9% NaCl)-treated blood microscope for any damage to RBCs. AM-NLopIG- and control (0.9% NaCl)-treated blood with AM-NLopIG. It was evaluated by treating with goat blood and observed under a samples were evaluated for shrinkage and swelling to the blood cells and did not show samples were evaluated for shrinkage and swelling to the blood cells and did not show microscope for any damage to RBCs. AM-NLopIG- and control (0.9% NaCl)-treated blood any damage in RBC, revealing that the formulation is isotonic (Figure 9A,B). The results samples were evaluated for shrinkage and swelling to the blood cells and did not show any any damage in RBC, revealing that the formulation is isotonic (Figure 9A,B). The results of the study are supported by the findings of the HET-CAM results. It also revealed no damage in RBC, revealing that the formulation is isotonic (Figure 9A,B). The results of the of the study are supported by the findings of the HET-CAM results. It also revealed no damage to the CAM. study are supported by the ﬁndings of the HET-CAM results. It also revealed no damage damage to the CAM. to the CAM. Figure 9. Isotonicity evaluation image of (A) AM-NLopIG and 0.9% (B) NaCl. 2.8.9. Antimicrobial Study The antimicrobial activity of AM-NLopIG and eye drop was determined by the cup plate method against S. aureus and E. coli (Figure 10). AM-NLopIG depicted ZOI of 17.5 ± Figure 9. Isotonicity evaluation image of (A) AM-NLopIG and 0.9% (B) NaCl. Figure 9. Isotonicity evaluation image of (A) AM-NLopIG and 0.9% (B) NaCl. 1.7 mm and 20.4 ± 2.1 mm against S. aureus and E. coli at 24 h. The same formulation was further evaluated at 48 h and there was a significant (p < 0.05) enhancement in ZOI. It 2.8.9. Antimicrobial Study 2.8.9. Antimicrobial Study The antimicrobial activity of AM-NLopIG and eye drop was determined by the cup plate method against S. aureus and E. coli (Figure 10). AM-NLopIG depicted ZOI of The antimicrobial activity of AM-NLopIG and eye drop was determined by the cup 17.5 1.7 mm and 20.4 2.1 mm against S. aureus and E. coli at 24 h. The same formulation plate method against S. aureus and E. coli (Figure 10). AM-NLopIG depicted ZOI of 17.5 ± was further evaluated at 48 h and there was a signiﬁcant (p < 0.05) enhancement in ZOI. It 1.7 mm and 20.4 ± 2.1 mm against S. aureus and E. coli at 24 h. The same formulation was showed a ZOI of 22.8 2.2 mm and 26.1 1.9 mm against S. aureus and E. coli at 48 h. The eye drop showed less antibacterial activity against S. aureus and E. coli as 19.1 1.4 mm further evaluated at 48 h and there was a significant (p < 0.05) enhancement in ZOI. It and 21.1 2.2 mm, respectively, at 24 h. Signiﬁcantly less activity was observed at 48 h. Gels 2022, 8, x FOR PEER REVIEW 12 of 18 showed a ZOI of 22.8 ± 2.2 mm and 26.1 ± 1.9 mm against S. aureus and E. coli at 48 h. The eye drop showed less antibacterial activity against S. aureus and E. coli as 19.1 ± 1.4 mm and 21.1 ± 2.2 mm, respectively, at 24 h. Significantly less activity was observed at 48 h. It Gels 2022, 8, 255 12 of 18 showed ZOI of 13.3 ± 1.2 mm and 15.8 ± 1.7 mm. The prepared formulation AM-NLopIG showed significantly (p < 0.05) higher susceptibility (1.4-fold and 1.5-fold) than the eye It showed ZOI of 13.3 1.2 mm and 15.8 1.7 mm. The prepared formulation AM- drop. The enhanced antibacterial activity is due to the nano-sized particles and high sur- NLopIG showed signiﬁcantly (p < 0.05) higher susceptibility (1.4-fold and 1.5-fold) than the face energy. It will have a large surface area to act on the cell wall of microorganisms and eye drop. The enhanced antibacterial activity is due to the nano-sized particles and high surface energy. It will have a large surface area to act on the cell wall of microorganisms and increase the membrane permeability of the bacteria due to more enhanced mucoadhesive increase the membrane permeability of the bacteria due to more enhanced mucoadhesive properties than the eye drop. properties than the eye drop. Figure 10. Antimicrobial graph of AM-NLopIG and eye drop evaluated against S. aureus and E. coli. Figure 10. Antimicrobial graph of AM-NLopIG and eye drop evaluated against S. aureus and E. coli. Results shown as mean SD. *** indicates signiﬁcant difference to the marketed eye drop. Results shown as mean ± SD. *** indicates significant difference to the marketed eye drop. 3. Conclusions An Azithromycin-loaded nano lipid carrier was prepared by the emulsiﬁcation– 3. Conclusions homogenization method. The formulations were optimized by BBD design using ho- An Azithromycin-loaded nano lipid carrier was prepared by the emulsification–ho- mogenization speed (A), surfactant concentration (B), and lipid concentration (C) as inde- pendent variables. The prepared formulations showed nano-metric size and high encap- mogenization method. The formulations were optimized by BBD design using homoge- sulation efﬁciency. The optimized formulation (AM-NLop) showed a nanoparticle size of nization speed (A), surfactant concentration (B), and lipid concentration (C) as independ- 154.1 6.35 nm, entrapment efﬁciency of 78.15 1.9%, a PDI value of 0.18, and negative zeta potential (34.24 mV). It was further converted into the sol-gel system to enhance the ent variables. The prepared formulations showed nano-metric size and high encapsula- mucoadhesion. It showed optimal physicochemical properties in the solution as well as in tion efficiency. The optimized formulation (AM-NLop) showed a nanoparticle size of gel form. The release and permeation study results depicted prolonged drug release as well 154.1 ± 6.35 nm, entrapment efficiency of 78.15 ± 1.9%, a PDI value of 0.18, and negative as enhanced permeation (1.82-fold higher than AM-IG and 2.61-fold higher than eye drop). AM-NLopIG was found to be isotonic as well as non-irritant (HET CAM and histopathol- zeta potential (−34.24 mV). It was further converted into the sol-gel system to enhance the ogy). The antibacterial study results revealed greater activity (1.4-fold and 1.5-fold) against mucoadhesion. It showed optimal physicochemical properties in the solution as well as both organisms. From the results, it can be concluded that the prepared AM-NLs and AM-NL-based in gel form. sol-gel The re system lease is an an ideal d perme deliveryasystem tion st to ud treat y res ocular ult diseases. s depicted prolonged drug release as well as enhanced permeation (1.82-fold higher than AM-IG and 2.61-fold higher than eye 4. Materials drop). AM-NLopIG was found to be isotonic as well as non-irritant (HET CAM and his- Azithromycin (AM) was procured from Alembic Pharmaceutical Ltd. (Ahmedabad, India). Glyceryl behenate (GB, melting point ~70 C), Labrasol, and Precirol ATO-5 were topathology). The antibacterial study results revealed greater activity (1.4-fold and 1.5- procured from Gattifosse (Mumbai, India). Tripalmitins, Stearic acid, Myristic acid, Glycerol fold) against both organisms. From the results, it can be concluded that the prepared AM- monostearate, Glyceryl monooleate, methanol, and acetonitrile were procured from Sigma- NLs and AM-NL-based sol-gel system is an ideal delivery system to treat ocular diseases. Aldrich (St. Louis, MO, USA). Isopropyl myristate, Miglyol, Coconut oil, Sunﬂower oil, Labrasol, and Sesame oil were procured from Loba Chemie (Mumbai, India). The Span 60, Span 20, Kolliphor EL, and Cremophor RH60 were procured from Acros organic (Somerset, 4. Materials NJ, USA). Zaha eye drops (Azithromycin eye solution, 1% w/v, Ajanta Pharma, India) were purchased from a local pharmacy. Azithromycin (AM) was procured from Alembic Pharmaceutical Ltd. (Ahmedabad, India). Glyceryl behenate (GB, melting point ~70 °C), Labrasol, and Precirol ATO-5 were procured from Gattifosse (Mumbai, India). Tripalmitins, Stearic acid, Myristic acid, Glyc- erol monostearate, Glyceryl monooleate, methanol, and acetonitrile were procured from Sigma-Aldrich (St Louis, MO, USA). Isopropyl myristate, Miglyol, Coconut oil, Sunflower oil, Labrasol, and Sesame oil were procured from Loba Chemie (Mumbai, India). The Span 60, Span 20, Kolliphor EL, and Cremophor RH60 were procured from Acros organic (Som- erset, NJ, USA). Zaha eye drops (Azithromycin eye solution, 1% w/v, Ajanta Pharma, In- dia) were purchased from a local pharmacy. 5. Experimental Gels 2022, 8, 255 13 of 18 5. Experimental 5.1. Screening of Lipids and Surfactant The screening of lipids was performed by the solubility of AM in the different solid lipids for the formulation of NLs. The excess amount of AM was added to each melted solid lipid (Glyceryl behenate, Tripalmitin (~64 C), Stearic acid (~69 C), Myristic acid (~52 C), Glycerol monostearate (~74 C), Glyceryl monooleate (~40 C), Precirol ATO-5 (~55 C)) in a glass vial. Similarly, the excess amount of AM was added to liquid lipids (Isopropyl myristate, Miglyol, Coconut oil, Sunﬂower oil, Labrasol, Sesame oil) and surfactants (Span 60, Span 20, Kolliphor EL, Cremophor RH60) in a glass vial. The samples were vortexed and kept in a water bath shaker (JULABO, Izumi Osaka, Japan) for 72 h. The mixture was centrifuged at 5000 rpm for 30 min, and then AM in each solid lipid was determined by a UV spectrophotometer (Genesys 10S UV-Vis, Thermo Scientiﬁc, Waltham, MA, USA) after appropriate dilution. 5.2. Selection of Solid and Liquid Lipid Ratio The melted solid and liquid lipids in different ratios (9:1, 8:2, 7:3, 6:4, 5:5, 2:8) were mixed with continuous stirring. The mixture was cooled, and the smear of lipids was prepared over filter paper. The separation of oil droplets over the filter paper was visually observed. 5.3. Optimization Design Expert software (Design Expert, version 8.0.6, State-Ease Inc., Minneapolis, MN, USA) was used for the optimization [39]. The purpose of this design was to examine the level of independent constraints, namely, homogenization speed (A, 12,000–20,000 rpm), surfactant concentration (B, 1.5–4.5%), and lipid concentration (1.5–4.5%), to achieve the desired size and entrapment efﬁciency. The polynomial Equation (3) was used to evaluate the statistical assessment of different independent variables on dependent constraints and can be given as: 2 2 2 Y = b + b A + b B + b C + b AB + b AC + b BC + b A + b B + b C (3) 0 1 2 3 12 13 23 11 22 33 where Y is the response related to each independent constraint; b is the intercept; b –b 0 1 33 are the regression coefﬁcients obtained for observed experimental values; and A, B, and C are the coded values of the independent factors. The coefﬁcients b , b , and b show 1 2 3 the individual parameter effect on the responses when two other parameters remain constant. The interaction terms (like b –b ) display the response modiﬁcation when the 12 23 two factors are simultaneously altered. Based on the preliminary screening, a range of various independent constraints were decided. These values were ﬁtted in the Design Expert software to obtain the composition of 17 formulations including ﬁve center points (common composition). The formulations were prepared, and their particle size and encapsulation efﬁciency data were placed in BBD to derive the predicted values, different polynomial equations, and model graphs. The obtained results were evaluated to examine the effects of independent constraints on dependent factors. 5.4. Development of Azithromycin-Loaded Nanostructure Lipid Carrier AM-NLs were developed by the emulsiﬁcation–homogenization technique as per a previously reported method with slight modiﬁcations [20]. The various batches (AM-NL1– AM-NL17) were prepared as per the given composition of Table 1. The solid lipid was melted at above 5 C of its melting point and the liquid lipid and was added with con- tinuous stirring to make the homogeneous mixture. The aqueous surfactant solution was prepared in distilled water and heated at the same temperature. The hot aqueous surfactant solution was added to the melted lipid mixture at 15,000 rpm for 15 min to form a coarse primary emulsion. The primary coarse emulsion was further subjected to a homogenizer Gels 2022, 8, 255 14 of 18 (T25 digital Ultra-Turrax IKA, Staufen, Germany) for 5 min. The prepared emulsion was cooled to room temperature to form AM-NLs and further stored for evaluation. 6. Characterization 6.1. Size, PDI, and Zeta Potential The size, PDI, and zeta potential were analyzed by a zeta sizer (NanoZS90, Malvern Instrument Ltd., Malvern, UK). The diluted NLs (100-fold) were placed in cuvettes, and size and PDI were determined. For zeta potential, the same diluted sample was placed in electrode-containing cuvettes, and then zeta potential was determined. 6.2. Entrapment Efﬁciency (% EE) The ultracentrifugation method was employed for the determination of EE from AM- NLs. AM-NLs were placed in a centrifugation tube and centrifuged at 15,000 rpm (Remi, cooling centrifuge, Mumbai, India) for 30 min. The supernatant was collected and further diluted to evaluate the AM content by a UV spectrophotometer, and % EE was calculated by the following equation: Total AM Free AM % EE = 100 (4) Total AM 6.3. X-ray Diffraction (XRD) Analysis An XRD instrument (Ultima IV, Rigaku Inc., Tokyo, Japan) was used to determine the crystallinity of AM after encapsulating into AM-NLop. Each sample was ﬁlled in an XRD sample holder and scanned between 5 and 60 at the 2-theta level. Diffractograms of both samples were recorded to compare the change in peak height and width. 6.4. Formulation of AM-NLop In Situ Gel The optimized formulation (AM-NL13) based on particle size and encapsulation efﬁciency was further converted into in situ gel by using mucoadhesive polymers. Weighed quantities of sodium alginate (1.5%, w/v) and hydroxyl propyl methyl cellulose (0.5%, w/v) were soaked overnight in distilled water. AM-NL13 was dispersed in polymeric solution to form in situ gel dispersion equivalent to 1% AM and pH was adjusted to 6.5. 6.5. Clarity and Gelling Ability The clarity and gelling ability of AM-NLs in situ gel (AM-NLopIG) were checked visually. For gelling strength, AM-NLopIG (50 L) was added to simulated tear ﬂuid (2 mL) in a vial and gelling time was noted [40]. 6.6. Viscosity Determination The viscosity of AM-NLopIG formulation in solution and gel form was measured by a Brookﬁeld viscometer (Fungi Lab, Madrid, Spain) using spindle number LV3 at 50 rpm angular speed. The temperature was ﬁxed at 37 0.5 C for the study. 6.7. Texture Analysis This study was performed using a texture analyzer (TA.XTplus100C, stable Microsys- tem, Ltd., Surrey GU7 1YL, Warrington, UK) as per the previous prescribed method [41]. AM-NLopIG was placed into the sample holder and pressed by a 20 mm diameter cylindri- cal probe (20 s interval, 2 mm depth). The hardness, cohesiveness, and adhesiveness were analyzed, and the results were compared with the eye drop. 6.8. In Vitro Drug Release The comparative release study of AM-NLopIG, AM-IG (control), and eye drop was performed using a dialysis bag (MWCO 12 kDa). The pretreated dialysis bag was ﬁlled with each formulation and dipped into STF (250 mL) at a temperature of 37 0.5 °C. At Gels 2022, 8, 255 15 of 18 a ﬁxed time, 2 mL release content was collected and the same volume of fresh STF was added to maintain the uniform release media volume. The released content was ﬁltered and further diluted to measure the absorbance using a UV spectrophotometer. 6.9. Trans-Corneal Permeation Study The study was performed using goat cornea collected from the slaughterhouse. The whole eyeball was collected and stored in NaCl (0.9%) at 4 C. The cornea was removed from the eyeball with the sclera and washed. Simulated tear ﬂuid (STF) was used as per- meation media and ﬁlled into the receptor compartment of the diffusion cell (area 0.6 cm , volume 10 mL). The cornea was mounted between the donor and acceptor compartment and the temperature was maintained at 37 0.5 C. AM-NLopIG, AM-IG, and eye drop (2 mg of the AM, in percentage) were ﬁlled into the donor compartment, and at a ﬁxed time point, the released content (1 mL) was removed. The blank fresh STF was replaced into a diffusion cell to keep a uniform volume. The collected release content was ﬁltered and further diluted for evaluation by the previously validated HPLC method [42]. The HPLC method was performed using mobile phase composition ammonium acetate buffer (0.05M and pH 8) and acetonitrile (60:40 v/v). The study was performed at a ﬂow rate of 1 mL/min with an injection volume of 20 L. The % permeation and ﬂux were calculated. Concentration Flux = (5) Permeation area ime Concentration Drug Permeation = dilution (6) applied area 6.10. Corneal Hydration The corneal hydration test was performed to evaluate the hydration of the cornea after the treatment with AM-NLopIG. The cornea was collected after the permeation study and the initial weight was noted (wet weight). The cornea was placed into the oven (Thermo Scientiﬁc, Osterode, Germany) at 80 C for drying. The cornea was removed from the oven and reweighed to note the dry weight. The % corneal hydration (CH) was calculated using the formula as reported by Mudgil et al. [43]. Wet weight Dry weight CH (%) = 100 (7) Dry weight 6.11. Histopathology Study The histopathology of the cornea was evaluated to check the internal damage after treatment with a prepared formulation. The cornea was collected after the permeation study, washed with STF, and stored in formalin solution (10% v/v.) The cornea was treated with 0.9% NaCl solution taken as a control. Each treated cornea was cleaned with alcohol and mounted with molten parafﬁn. The cross-section of the cornea was cut and stained with hematoxylin and eosin to evaluate under a high-resolution microscope (BA210m Motic microscope, Selangor Darul Ehsan, Malaysia). 6.12. HET-CAM Irritation Study The in vitro irritation AM-NLopIG, negative control (0.9% NaCl), and positive con- trol (sodium lauryl sulphate, 1% w/v) were evaluated by using hen egg chorioallantoic membrane (HET-CAM). The fertilized hen eggs were procured from a poultry farm and incubated for 10 days in a humidity-controlled incubator at 37 0.5 C/5% RH. On the 10th day, eggs were collected and shells were removed from the air chamber side. The inner membrane was wetted by adding a drop of 0.9% NaCl and then carefully removed by using forceps to visualize developed CAM. Then, 2–3 drops of the AM-NLopIG, NaCl (0.9% w/v), and sodium lauryl sulphate (1% w/v) were added over CAM and observed for 5 min to check for damage. The scores were given as per the standard scoring scale at Gels 2022, 8, 255 16 of 18 different time points, i.e., 0–0.9 for non-irritants, 1–4.9 for weak irritants, 5–8.9 for moderate irritants, and 9–21 for severe irritants [44]. 6.13. Antimicrobial Study The activity of AM-NLopIG and eye drop was evaluated by the cup plate method against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The required quantity of nutrient agar media was weighed, transferred into a conical ﬂask, and dissolved in distilled water. The media was sterilized by autoclave at 121 C for 15 min. The micro-organisms S. aureus and E. coli (0.1 mL) were mixed with nutrient agar media and transferred to a sterilized Petri plate under aseptic conditions. The media was kept for solidiﬁcation in an aseptic area. Using a sterilized borer, 5 mm wells were created and the samples (AM-NLopIG and eye drop) were added to evaluate their efﬁcacy. The Petri plates were kept for 1 h and further incubated at 37 C for 24 h and 48 h to evaluate the zone of inhibition (ZOI). Author Contributions: S.J.G. and M.N.b.J.—Conceptulization, Resources and Funding; A.Z. and S.S.I.—Experi-mental work and project administration; M.Y. and M.K.—software and data curation; S.A.—Supervision, review and editing; M.M.G. and F.M.A.—Data analysis and writing original draft. All authors have read and agreed to the published version of the manuscript. Funding: Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R108), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Not Applicable. Acknowledgments: This research project is supported by Princess Nourah bint Abdulrahman Univer- sity Researchers Supporting Project number (PNURSP2022R108), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Tangri, P.; Khurana, S. Basics of ocular drug delivery systems. Int. J. Res. Pharm. Biomed. Sci. 2011, 2, 1541–1552. 2. Wafa, H.G.; Essa, E.A.; El-Sisi, A.E.; El Maghraby, G.M. Ocular ﬁlms versus ﬁlm-forming liquid systems for enhanced ocular drug delivery. Drug Deliv. Transl. Res. 2021, 11, 1084–1095. [CrossRef] [PubMed] 3. Gilhotra, R.M.; Nagpal, K.; Mishra, D.N. 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Gels – Multidisciplinary Digital Publishing Institute

Published: Apr 21, 2022

Keywords: NLs; in situ gel; ocular delivery; azithromycin; antibacterial; HET CAM

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Formulation and Evaluation of Nano Lipid Carrier-Based Ocular Gel System: Optimization to Antibacterial Activity

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