INTRODUCTION:

Fungal eye infections such as fungal keratitis represent a widespread sight-threatening ocular disease that might lead eventually to corneal blindness¹. Candida species are regarded as the major cause of fungal keratitis causing Candidiasis, a very common type of superficial fungal infections².

For the treatment of this type of infection, topical application of ophthalmic drugs in the form of eye drops is considered the most efficient and preferred route for drug delivery³. These eye drops represent a painless and simple method of drug application, thus achieving patient convenience allowing a localized effect of the administered drug with minimal chance for systemic absorption⁴ . However, the special physiology, anatomy, and biochemistry of the eye render this organ difficultly accessed by foreign compounds in addition to the lipophilic nature of most antifungal agents which together results in poor ocular bioavailability^5,6.

Miconazole nitrate (MN); is a broad-spectrum antifungal agent of the imidazole group, act through the inhibition of ergosterol biosynthesis causing the lysis of the fungal cell membrane and peroxidase inhibition, leading to the accumulation of peroxide within the cell and eventually cell death⁷. MN is well known for its powerful activity, especially against the Candida species, the common cause for fungal keratitis⁸, and simultaneously relatively low toxicity in the ocular tissue⁹. However, its use topically is hindered by its very low solubility which lowers its efficacy and achievement of optimum bioavailability in the eye¹⁰. In addition, its inability to penetrate the ocular tissue due to its hydrophobic nature deters its penetration into the eye tissue all of which hinders its use as an ocular antifungal despite its effectiveness⁶.

Thus, to overcome some of the previously mentioned constraints, nanocarrier-based formulations offer a promising strategy to enhance the solubility and ocular bioavailability of poorly water-soluble drugs^11,12 . Among the various investigated nanocarriers, polymeric micelles have acquired much attention as a potential candidate for the ocular application of active agents¹³. Polymeric micelles are colloidal carriers produced by the self-assembling of graft or block amphiphilic copolymers^14,15. They offer several favorable characteristics for successful ocular delivery, including good ocular tolerance, the ability to incorporate both hydrophobic and hydrophilic drugs and control their release pattern¹⁶. The small size of these micelles can also promote corneal absorption through the facilitated cellular uptake of the drug by endocytosis and thus improving drug permeability across the corneal epithelium³.

The block copolymers of poly (ethylene oxide) - poly (propylene oxide) - poly (ethylene oxide) (PEO-PPO-PEO) are considered one of the most explored amphiphilic substances¹⁷. These polymers act as non-ionic surfactants capable of forming core-shell micelles composed of a hydrophilic shell of PEO which maintains stability during dispersion, enclosing a hydrophobic core of PPO serving as a suitable environment for the incorporation of hydrophobic substances, above the critical micelle concentration (CMC)¹⁸. Two common families are commercially available; the linear and bifunctional PEO–PPO–PEO triblocks named pluronic or poloxamers and their branched four-arm analog known as tetronic or poloxamine¹⁹. Both types are characterized by their biocompatibility, reduced toxicity and are readily available in variable ratios of ethylene oxide/propylene oxide and different molecular weights²⁰. However, they have been reported to suffer poor drug encapsulation for their micelles and requiring a high drug to polymer ratio to achieve adequate drug encapsulation efficiency²¹.

Solupus^®(SP) is a polyvinyl caprolactam-polyvinyl acetate-poly- ethylene glycol graft copolymer with amphiphilic properties and can form micelles when its concentration is higher than its CMC, thereby enhancing the solubility of poorly water-soluble drugs²². However, the formed micellar systems of SP suffer from poor stability which in turn hinders their use²³. Mixed micellar systems of poloxamer and poloxamine with SP can offer combined benefits in enhancing the encapsulation efficiency with increased stability²⁴. It is worth noting that MN ocular mixed micelles were previously attempted by researchers, however, the safety and stability of the prepared micelles were not reported, which is a major concern with such formulations²⁵.

Based on the aforementioned considerations, the present work aims to explore the potential of polymeric nano-micelles for the ocular delivery of MN using three types of block copolymers: Pluronic F127 (F127), Pluronic P123 (P123), and Tetronic T701 (T701). The in vitro properties of single polymers and their mixtures were studied to establish the optimal formulation of MN-loaded nano-micelles for incorporation with SP to further enhance its release and encapsulation properties. The final prepared SP incorporated nano-micellar systems were subjected for in vitro characterization as well as in vivo antifungal efficacy of the selected system was evaluated against C. albicans in the treatment of induced ocular candidiasis using rabbits as model animals.

MATERIAL AND METHODS:

Chemicals and reagents:

Miconazole nitrate was kindly gifted by EVA Pharma, Egypt. Tetronic^® T701 (T701), Pluronic^® P 123 (P123), and Pluronic^® F127 (P123) were purchased from Sigma- Aldrich (Germany). Soluplus^® was obtained from BASF SE, Germany. All other chemicals were of the highest available commercial grade and purchased from El-Nasr Chemicals (Cairo, Egypt). Male albino rabbits weighing 2-2.5 kg were obtained and kept in the animal house of the National Research Centre (NRC) (Cairo, Egypt) under conventional laboratory conditions.

Design of experiment:

A 3³ full factorial design was done using Minitab^®version 17.1.0 (Minitab Inc., State College, PA, USA), by varying the concentrations of P123, F127, and T701 in different ratios (1:1, 2:1, and 1:2) so that the sum of excipients in each formulation was kept constant at 1g (10% W/V). The resultant nine formulas are present (MPM-1 to MPM9 in Table (1)). For comparison purposes, single formulations of the above polymers were prepared (S-F127, S-P123 and, S-T701).

Table (1): Composition of MN-loaded SPM and MPM systems and their respective results of %EE (n=3), particle size (n=3), CP (n=3), and %Q₆ (=6)

Formulation code*	Polymer (%w/v)			%EE	Particle size (nm)	PDI	CP(°C)	%Q₆
Formulation code*	P123	T701	F127	%EE	Particle size (nm)	PDI	CP(°C)	%Q₆
S-P123	10	-	-	20.69±5.65	21.61 ± 1.48	0.106	>100	60.31±6.64
S-T701	-	10	-	34.57±3.71	322.86±15.71	0.361	28.11± 1.18	45.02±4.12
S-F127	-	-	10	12.65±2.21	76.65 ± 3.54	0.284	95±2.45	65.38±7.12
MPM1	6.67	3.33	-	29.11±4.49	124.1 ± 4.16	0.138	46.95±2.12	58.99±2.95
MPM2	5	5	-	31.49±2.75	123.6 ± 4.03	0.195	35.90±1.44	52.72±7.68
MPM3	3.33	6.67	-	40.98±5.52	183.17±5.61	0.174	38.40±0.566	48.86±4.66
MPM4	-	6.67	3.33	35.73±1.13	283.40±4.24	0.122	39.52±0.354	43.19±2.46
MPM5	-	5	5	29.17±3.91	270.95±7.41	0.312	41.10±0.424	54.18±6.18
MPM6	-	3.33	6.67	23.97±1.01	255.85±2.12	0.287	46.92±0.594	59.01±1.89
MPM7	3.33	-	6.67	29.46±3.54	40.54 ± 3.11	0.108	> 100	64.17±2.01
MPM8	5	-	5	32.53±5.23	34.39 ± 1.48	0.117	> 100	67.33±5.23
MPM9	6.67	-	3.33	35.12±3.18	37.88 ± 2.64	0.192	> 100	68.82±6.48

*Theoretical concentration of MN was 0.2% w/v in all the formulas.

For analysis and optimization of the model, the monitored responses were particle size (PS), cloud point (CP), encapsulation efficiency (%EE), and %released at 6 hours (%Q₆). Data collected for each response of the dependent variables were analyzed by fitting the raw responses into a first-order multiple linear regression model and the adjusted multiple correlation coefficient (adjusted R2) and the p values for the overall F-ratio test were compared and analyzed²⁶. Analysis of variance (ANOVA) was used to evaluate the statistical significance of the model at a level of significance (α) of 0.05²⁷. For the selection of the optimized nano-micellar system for future work, Desirability index (D) values were utilized which reflects the level of satisfaction with a given combination of independent variables with a D value of 0 indicating a completely undesirable formula and a value of 1 indicating a completely desirable one²⁸. In this study, the optimized nano-micellar system was regarded as the formula with the highest D value and the selection criteria was achieving the smallest particle size with the highest %EE, CP, and (%Q₆). The four criteria were considered equally important; therefore, the weights were set to 1/4.

Preparation of single (SPM) and mixed polymeric nano-micellar systems (MPM):

The direct equilibrium technique was used for the preparation of the MPM nano-micellar systems as well as for the SPM systems²⁹. For the preparation of the SPM, T701, P123, and F127 (10%w/v) were dissolved in distilled water and the systems were then kept equilibrating for 24 hrs at 25°C to help micelle formation. The same method was used for the preparation of the MPM, T701: P123, T701: P127, and P123: F127 (10% w/v) with ratios of 1:1, 2:1, and 1:2 was first allowed to dissolve in distilled water to constitute a total concentration of (10% w/v). Then, a known excess amount of MN (0.2 %w/v) was added to this system and shaken for 48 hours. Afterward, the undissolved drug was removed by centrifugation at 12,000rpm for 15 min (Union 32R, Hanil, Korea).

Characterization of prepared SPM and MPM:

Particle size:

The mean micellar size and size distribution in terms of polydispersity index (PDI) of MN-loaded SPM and MPM formulas were determined using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The measurements were performed at 25℃ and a scattering angle of 90º after appropriate dilution of the samples with distilled water.

Encapsulation Efficiency (%EE)

The prepared nano-micellar systems were centrifuged at 12000 rpm for 60 min at 4°C (Union 32R, Hanil, Korea). Then, the residue was diluted with ethyl alcohol and sonicated for 30 min to disrupt the micellar structure. The encapsulated amount of MN was assessed spectrophotometrically at 271nm (Shimadzu UV spectrophotometer, 2401/PC, Japan) ³⁰ . The encapsulation efficiency was calculated according to the following equation:

Actual MN Amount

% EE = --------------------------------- X 100 (1)

Theoretical MN Amount

Cloud point (CP):

The prepared systems were added to 20 mL glass tubes then immersed in a water bath at 25°C. The temperature increased gradually at a rate of 1°C/min till the first visual appearance of turbidity which was recorded as the CP³¹.

In vitro release study:

The release pattern of MN from the prepared nano-micellar systems was studied using the dialysis bag diffusion technique (Dialysis tubing cellulose membrane, Sigma Co., USA; Molecular weight cut-off 12,000 –14,000). 2mL from each formulation as well as free MN suspension were added to the dialysis bag, which was then sealed at both ends to avoid leakage. The bag was immersed in screw-capped glass bottles filled with 100mL release medium (phosphate buffer pH 7.4³²) and shaken at 100rpm in a thermostatically controlled shaking incubator (GFL 3203, Germany) at 37±0.5°C. At specific time intervals, 2 mL samples were withdrawn, and the same volume of fresh release medium was added to keep a constant volume. The samples were then analyzed for the amount of MN released spectrophotometrically at 271nm and the cumulative percentages of drug released were calculated. The experiment was performed in triplicate and the data were expressed as mean values ± S.D.

Solubility study of MN using Soluplus^® (SP):

An excess amount of MN was added to 10mL aqueous solutions of different concentrations of SP namely (0.5, 1, and 1.5 %w/v) as well as distilled water to check the initial solubility of MN. The vials were sealed and shaken using a thermostatically controlled shaker for 48 hrs. at 37±0.5°C after which the samples were filtered using 0.45µm filter and the amount of MN in the filtrate was determined spectrophotometrically at λmax 271nm (n=3).

Preparation and characterization of MN-loaded SP-mixed nano-micelles (SP-MPM):

Optimized mixed nano-micellar system MPM9 was prepared as described previously then SP (1.5% w/v) was dissolved in distilled water and was added to the prepared formulas. The systems were then kept equilibrating for 24 hrs. at 25°C then the preparation proceeded as previously described and the resultant formulas were assessed as formerly established for particle size, %EE, CP, and in vitro release study.

Differential scanning calorimetry (DSC):

The thermograms of MN, SP, F127, and MN-loaded SP-MPM were recorded utilizing a differential scanning calorimeter (Shimadzu, DSC-60. Japan). A sample (2–4 mg) of powder was accurately weighed into standard aluminum pans using an empty pan as a reference. The samples were heated and scanned at a rate of 10 °C/min over a temperature range of 20 –350 °C under liquid nitrogen (25 mL/min).

X-ray powder diffraction (XRD):

X-ray powder diffraction patterns of MN, SP, F127, and MN-loaded SP-MPM were evaluated using an X-ray diffractometer (Scintag Inc., USA). Samples were irradiated using Ni filtered, CuKa radiation at a voltage of 45 kV, and a 9 mA current. The diffraction angle (2 θ) ranged from 0 to 90° at a scanning rate of 1° min− 1.

Transmission electron microscopy (TEM):

The morphology of MN-loaded SP-MPM was examined using TEM (JEOL Co., JEM-2100, Japan). One drop of the diluted sample was placed onto a carbon-coated copper grid and stained with 0.2% (w/v) phosphotungstic acid for 30 s then left to dry for 10 min at room temperature. The samples were examined, and the micrographs were taken at suitable magnification power.

Stability Study:

The selected formula (SP-MPM) was stored in amber glass vials under refrigeration (4 ± 2°C) for 3 months. Physical appearance, %EE and particle size were measured and compared with the freshly prepared formula.

In vivo pharmacodynamics study:

Animals:

Adult male albino rabbits weighing 2-2.5 kg were kept in individual cages under well-defined and standardized conditions (humidity and temperature-controlled room; 12 h light and 12 h dark cycle). Animals were fed with standard dry food and water ad libitum. The eyes were first inspected with a slit lamp and those with no sign of any inflammation were included in this study. All animal experiments were performed according to the protocol approved by the Institutional Animal Ethics Committee (Medical Research Ethics Committee (MREC) of the NRC, Cairo, Egypt.

Susceptibility test:

The test was performed for the selected nano-micellar system as well as MN suspension (0.2% w/v). After a single installation (30 mL) of the formulation, each in the conjunctival sac of the right eye of three rabbits, sterile filter paper discs (6 mm diameter, Whatman no. 1) were located for 30 seconds at specific time intervals under the eyelid of each rabbit namely 30 mins, 1,2,3,4,5,6, 8 and 12 hours. The discs were added to the inoculated Sabouraud dextrose broth (SDB) tubes, and then the tubes were incubated for 24 h at 37±0.5℃. The inhibition of fungal growth was assessed by measuring the culture's optical density spectrophotometrically (Shimadzu, UV- 2401 PC, Japan) at 600 nm. The level of MN in the eye tears after the ocular instillation of the tested formulation was determined as the percentage of inhibition calculated using the SDB medium inoculated with C. albicans NRRL Y-477 as control³³. The percentage inhibition was used as an indication of MN amount in tear fluid and was used to calculate the pharmacokinetic parameters of MN in eye tears using Microsoft Excel^® software. The area under the curve from 0 to 12 h (AUC_{0–12 h)}was estimated by the linear trapezoidal method to compare the antifungal effect of the tested nano-micellar formulation as well as MN suspension.

Induction and treatment of ocular candidiasis:

For candidiasis induction, a fungal suspension of C. albicans was adjusted to contain 133 CFU/mL with sterile physiological saline and each rabbit was infected with 100mL C. albicans suspension. The rabbits were randomly divided among four groups each containing three rabbits. Group 1 served as positive untreated control, group 2 received 0.2% MN suspension, Group 3 was treated with SP mixed nano-micelles SP-MPM, and Group 4 served as a negative control, and candidiasis was not induced. After 48 h of infection; candidiasis was confirmed through the appearance of redness, inflammation, and excessive tearing. Afterwards, treatment was started as a single instillation (30 mL containing 0.2% MN) every 12 h for 7 days while the control group did not receive any treatment. Swabs were taken from each infected area into sterile tubes containing 5mL of potato dextrose agar (PDB) during the five days of treatment. Serial dilutions were done and incubated for 24 h at 37°C after which the colonies were counted, and the percentage of fungal infection inhibition was determined.

Histopathological examination:

At the end of the experiment, the rabbit eyes were excised and processed for histopathological evaluation. The samples were fixed in 10% formalin solution for 48 h. The sections were cut from the paraffin block by microtome, stained with hematoxylin and eosin, then observed for any histopathological changes using a light microscope (Leica, DM-6000, Wetzlar, Germany) with an integrated camera.

Statistical analysis:

The obtained data were expressed as mean±SD and statistical analysis of the results was done using Minitab^®version 17.1.0 (Minitab Inc., State College, PA, USA). The significance of differences was determined by one-way analysis of variance (ANOVA) followed by the least significant difference test. Values of P < 0.05 were considered statistically significant.

RESULTS:

Particle size:

The particle size of prepared nano-micellar systems showed unimodal size distributions with small PDI-values ranging between 0.106 and 0.384 confirming the homogeneous distribution of the formed nano-micelles. As depicted from the results in Table (1), the average micelle size varied depending on the used polymer. T701 single micelle systems showed the largest particle size (322.86±15.71nm) compared to P123 or F127. F127 single micelles showed slightly larger micelles (76.65± 3.54nm) than that of P123 (21.61±1.48 nm). In the case of the mixed nano-micellar systems, P123/F127 (MMP7 to MMP9) mixed nano-micelles revealed narrow size distribution with average size varying from 34.39± 1.48 to 40.54± 3.11 nm. The addition of the more hydrophilic polymers (P123 and F127) to T701 produced a significantly smaller particle size ranging from 124.12± 4.16 nm to 183.17±5.61nm and 255.85±2.12nm to 283.40 ± 4.24 nm for T701:P123 (MMP1-MMP3) and T701:F127 (MMP4-MMP6) respectively compared to the single T701 system.

Encapsulation efficiency (%EE):

As shown in Table 2, all prepared systems produced a significant increase in MN solubility ranging from 12.65±2.21% to 40.98±5.52% compared to MN aqueous solubility (61.68±1.15µg/ml, 3.084% w/v) resulting in a subsequent increase in the amount of drug encapsulated. Regarding the single nano-micellar systems, T701 and P123 revealed much better solubilization capacity compared to F127 where the encapsulation ability of the used polymer was in the following order: T701 ≥ P123 > F127. Similarly, all mixed nano-micellar systems resulted in a significant increase in drug solubility (p<0.05), this increment varied according to the used copolymer and its ratio. T701 MPMs displayed a statistically significant higher %EE (p < 0.05) upon the addition of P123 or F127 where the higher the content of T701, the better enhancement of the encapsulation extent observed. T701/ p123: 2:1 (MMP3) produced a higher %EE (40.98±5.52%). However, a further increase in the P123 ratio resulted in a decline in the %EE as shown in MMP1 (29.11±4.49%). The addition of F127 to T701 at ratios 2:1 (MMP6) and 1:1 (MMP5) produced a subsequent decrease in %EE (23.97±1.01 and 29.17±3.91% respectively) compared to T701/ F127: 2:1 (35.73±1.13) Mixing of P123 and F127 (MMP7-MMP9) resulted in increasing %EE of the prepared mixed nano-micelles compared to single micellar systems, where increasing P123 ratio in the mixtures increase the solubilization of MN, with the highest encapsulation, was recorded for MMP 9 with a ratio of P123/F127: 2:1 (35.12±3.18%).

Cloud point (CP):

As presented in Table 1, T701 single micelles displayed phase separation at low temperature (28.11±1.18℃) compared to the more hydrophilic pluronics; P123 and F127 (>100 and 95±2.45℃, respectively). Mixtures of T701/P123 (MMP1-MMP3) and T701/F127 (MMP4-MMP6) exhibited lower CP values than the single Pluronic systems while P123/F127 (MMP7-MMP9) mixed systems demonstrated CP at temperatures >100℃.

In vitro release study:

The cumulative release profiles of MN from the prepared nano-micelles systems compared to the free drug are presented in Fig. 1A. As illustrated, MN exhibited a very low release profile with a %Q₆ of 21.23±2.46%. Meanwhile, %Q₆ from the different SPM and MPM formulas was found to be in the range of 43.19±2.46% to 68.99±2.95% exhibiting a sustained drug release profile. To determine the optimized formula for future work in this research, the formula with the maximum %EE, minimum PS, maximum CP and maximum %Q₆ was determined to be MPM-9 comprising of 3.33% (w/v) of F127 and 6.67%(w/v) of P123 total polymer concentration (desirability value=0.842). Thus, MPM9 will be further characterized in vitro and in vivo.

Preparation and characterization of MN-loaded SP-MPM:

Incorporation of SP (SP-MN) resulted in an increase in solubility of MN in water from 61.68±1.15µg/ml to 304.78±2.68 µg/ml, 538.84±5.79 µg/ml and 1373.33±3.33 µg/ml for concentrations 0.5%w/v, 1%w/v, and 1.5%w/v respectively, However, SP systems suffered from poor stability in room temperature evident a cloud point at 35.58±2.79°C (SP-MN). Thus, SP was incorporated in optimized mixed micelles formula MPM9 in a concentration of 1.5% w/v as it showed the highest solubilization capacity of MN (SP-MPM).

As shown in table (2), the addition of SP to the prepared SPM and MPM has resulted in a sharp and significant (p<0.05) increase in %EE of the optimized formula MPM-9 with an SP-MPM system achieving an approximate 3 fold increase from 35.12±3.18% to 99.10±7.03%. The stability of SP micellar systems was drastically improved by mixing with F127 and P123 as the cloud point increased from 35.58±2.79°C (SP-MN) to 76.95± 2.12°C (SP-MPM). As shown in Fig. 1B and Table 2, MN release from SP-MPM systems has also drastically improved with %Q₆ of 83.48±3.79% compared to SP-MN alone which increased %Q₆ to only 64.04±2.15%.

Differential scanning calorimetry (DSC):

DSC thermograms of MN, F127, SP, and SP-MPM are presented in Fig. 2A, MN and F127 showed a single endothermal peak at 85.2 and 58.72°C, respectively, corresponding to their melting points³⁴. P123 was not examined due to its semi-solid nature. The thermograms of SP-MPM did not show any peak of MN, with the presence of the characteristic peak of F127.

X-ray diffraction (XRD):

X-ray diffraction patterns of MN, F127, SP, and SP-MPM are illustrated in Fig.2B As observed, MN displayed intense sharp peaks in the 2θ range of 10-30°, while the X-ray diffractogram of pure F127 showed distinct peaks at 18.89 and 23.09⁰ indicating their crystalline nature³⁴. The diffraction spectra SP-MPM revealed the disappearance of the drug characteristic peaks with the presence of reduced peaks corresponding to F127.

Fig. 2: (A) DSC thermograms and (B) XRD diffractograms of MN, F127, SP, and MN-loaded SP-MPM nano-micelles formula.

Transmission electron microscopy (TEM) analysis

TEM micrographs of SP-MPM are presented in Fig. 3 As observed; the nano-micelles appeared as uniform spherical shaped, non-aggregated particles with narrow size distribution which conform with the measured particle size.

Fig. 3: TEM images of MN-loaded SP-MPM nano-micelles formula.

Stability Study:

MN-loaded SP-MPM has shown stability over the tested attributes with insignificant changes after storage for three months. The micellar system retained its physical appearance. The %EE changed from 99.19±7.03% of the fresh sample to 96.23±5.03% for the stored sample which was within the 5% limit specified by the ICH guideline (Q1A (R2)³⁵. Furthermore, the micellar system has shown an insignificant (p>0.05) change in particle size from 44.39±2.68 nm to 49.16±1.51nm as well as an insignificant change (p>0.05) in CP from 76.95±2.12°C to 74.21± 3.57°C thereby confirming the preservation of micellar system characteristics and its the inherent stability.

In vivo study:

Susceptibility test:

Fig. 4A depicts a significantly higher percentage inhibition of bacterial growth with SP-MPM formula which sustained its inhibitory effect at 91.55±3.46% for six hours before it started to decrease gradually to reach 51.85±1.86% at 12 hours, unlike MN suspension which its inhibition effect gradually decreased from the start of the experiment to reach a minimum of 8.18±1.21% at 5 hours. Moreover, The AUC _(0-12h) from the MN-loaded SP-MPM formula was calculated at 1584.73±2.21 which was significantly higher (p< 0.05) compared to MN suspension (259.04±2.92) which confirms the higher antifungal activity of SP-MPM compared to that obtained from MN suspension.

Induction and treatment of ocular candidiasis:

As shown in Fig. 4B, with SP-MPM mixed micellar formula was able to successfully treat the corneal keratitis as well as reducing the number of viable CFU recovered thereby reducing the signs of infection. This effect was significantly different from groups treated with MN Suspension which showed a higher CFU count. Both groups showed a significant difference between the infected and untreated control group (p<0.05).

Histopathological examination:

The untreated control group (Fig.5B) showed major histological alterations after 7 days from induction of infection, these changes were associated with massive inflammatory cells infiltration in the stroma of the eyelid with thickening in the inner cellular layer of the cornea in addition to congestion in the blood vessels of sclera and vacuole degeneration was detected in the retina compared to normal group (Fig. 5A). While the histopathological sections of the rabbits’ eyes after treatment with MN suspension for 7 days showed focal inflammatory cells aggregations in the inner and outer peripheral areas of the eyelid and vacuolization and degeneration in the outer and inner layer of the retina (Fig. 5C). On the contrary, the rabbits treated with MN-loaded SP-MPM manifested marked attenuation of observed inflammation, as no histopathological alterations were detected in all studied eye sections (Fig. 5D) including the retina and their return to normal conditions.

Fig. 4: (A) Percentage inhibition of C. albicans growth produced by SP-MPM nano-micelles formula compared to MN suspension in the eyes of albino rabbits throughout 12 hours (n=3). (B)Corneal colony counts for groups infected with C. albicans throughout 7 days of treatment with MN suspension and SP-MPM nano-micelles formula compared to the control group with no treatment (n=3).

Fig. 5: Histopathological examination for (A) negative control normal rabbit eyes, (B) untreated group infected with C. albicans (C) group treated with MN suspension, and (D) group treated with SP-MPM nano-micelles formula after 7 days of treatment.

DISCUSSION:

Currently, mixed nano-micellar systems have shown noticeable superiority to the single components revealing apparent synergistic characteristics concerning the nano-micellar stability and the enhanced drug encapsulation ³⁶. Thus, in this work, two linear Pluronics (F127 and P123) having the very close length of hydrophobic block P123 (PPO: 69) and F127 (PPO: 64) with different HLB values (22 and 8, respectively), in addition to tetronic 701 (T701) with different architecture, PPO (14) and HLB (1-7) have been utilized, investigating their potential for the formation of nano-micellar systems of the hydrophobic drug MN. A concentration of 0.2% w/v (0.2mg/ml) of MN was chosen as it is greatly higher than its reported minimum inhibitory concentration of 3.125µg/ml against C.albicans, thus ensuring its efficacy in vivo³⁷.

The particle size of nano-micellar systems is of extreme importance in ocular delivery to the eye as it relies on endocytosis and particle size-dependent paracellular transport, thus the smaller the particle size, the better the permeation of the nano-micellar system across the cornea and avoids drug loss due to reflex eye blinking and tear washing³⁸. Micellar size varied depending on the used polymer with T701 showing the largest particle size compared to P123 or F127 attributed to their polar and semi-polar nature enabling them to form small micelles in water³⁹. However, F127 showed slightly larger micelles than that of P123 due to the longer hydrophilic chain length (PEO) of F127 compared to P123⁴⁰. The addition of the more hydrophilic polymers (P123 and F127) to T701 produced a significantly smaller particle size compared to the single T701 system attributed to the hydrophobic interactions of the long PO block of T701 which tends to aggregate hindering the formation of stable nano-micelles in aqueous media. The presence of pluronics of higher HLB values would increase the kinetic stability of the nano-micelles ⁴¹ and resulted in gradual shrinkage of the micellar size leading to the production of smaller and more stable spherical nano-micelles capable of stabilizing and regulating the hydrophobic PO blocks of T701. This is manifested in the fast separation of non-ionic surfactant micellar systems into two phases upon heating to a threshold temperature, known as the CP. This property can be utilized as an indication of stability as higher CP indicates a more stable micellar system⁴². In the case of PEO/PPO (P123 and F127) copolymer solutions, the CP depends primarily on the copolymer composition showing higher CP values for copolymers with a large number of PEO units. These copolymers reveal a hydrophilic character with improved solubility in aqueous media and undergo phase separation at elevated temperatures. On the contrary, copolymers with shorter PEO blocks such as T701 generally exhibit poor water solubility and undergo micellization at relatively lower temperatures⁴³.

The solubilization capacity of micellar systems for poorly soluble drug is directly related to the compatibility between the micelle core and the drug ⁴⁴ with the solubility of the active compounds depending mainly on the hydrophobicity of the copolymer used in addition to the length of the hydrophilic block⁴⁰. As such, T701 being the most hydrophobic, showed the highest solubilization potential compared to P123 which has moderate hydrophobicity and the least was the hydrophilic pluronic F127 which has the longest hydrophilic chain length of polyoxyethylene as well⁴⁵. By forming mixed nano-micellar systems, A significant change in drug solubilization was observed (p<0.05) and varied according to the used copolymer and its ratio. T701 mixed nano-micelles displayed a statistically significant higher encapsulation capacity (p < 0.05) upon the addition of P123 or F127 where the higher the content of T701, the better enhancement of the %EE observed. However, further increase in the P123 ratio resulted in a decline in the %EE attributed to the retardation in the micelle stability by excessive stacking of cylindrical aggregates formed by P123 ⁴⁵. Similarly, the addition of F127 to T701 produced a subsequent decrease in %EE. Surprisingly, mixing of P123 and F127 (MMP7-MMP9) resulted in increasing %EE of the prepared mixed nano-micelles compared to single micellar systems, where increasing P123 ratio in the mixtures increase the solubilization of MN, with the highest encapsulation, was recorded for MMP9 with a ratio of 2:1 probably as F127 is more hydrophilic with a longer chain which likely prevented stacking of cylindrical P123 aggregates thereby achieving better encapsulation properties⁴⁶

MN, being a poorly water-soluble drug, naturally exhibited a low release profile. Nevertheless, MPM systems were able to counteract this problem. The superiority of the release profile of F127 and P123 can be attributed to their hydrophilicity which promoted the distribution of water molecules into the core of the micelles, resulting in the formation of more hydrophilic channels enhancing the drug release ⁴⁵. On the contrary, the more hydrophobic T701 retarded the diffusion of water into the core hindering the diffusion of MN leading to the observed slow-release profile. Moreover, the mixed micelles revealed a release pattern that was dependent on the type as well as the ratio of the involved copolymer. The presence of F127 and P123 resulted in a significant enhancement in the %MN released from the mixed micellar systems while T701 containing systems showed a slower release pattern owing to the increased interaction between T701 and hydrophobic drug.

In an attempt to further enhance the encapsulation and release characteristics of the prepared micellar systems, SP was incorporated in the optimized formula (MPM9) which comprised F127 and P123 in a ratio of 2:1. SP is a well-established solubilizing agent for insoluble drugs with a moderate HLB of 16, which can form micellar systems capable of encapsulating hydrophobic drugs as it is an amphiphilic moiety able to form micelles at very low concentrations and thus having a high solubilization power^22,
47. This is evident by the increase in solubility of MN upon the addition of SP. However, SP systems have been previously reported to suffer from poor stability in room temperature ²³ and confirmed by low CP of MN-loaded SP system. As such, SP-pluronic mixed micellar systems can combine the benefits of both systems. On one hand, SP can enhance the %EE due to its high solubilization power. On the other hand, F127 and P123 can enhance the stability of SP systems combined with increased hydrophobicity to aid in the permeation of the formula into the ocular tissue of the eye. SP-pluronic micellar systems were also reported to form in-situ gelling in the eye thus can retain the formulation in the eye⁴⁷.

Thus, SP was incorporated in the optimized MPM (MPM-9) to further increase the %EE of MN as well as to benefit from the stability and small particle size. SP incorporation did not affect the small particle size, however, has resulted in a sharp and significant (p<0.05) increase in %EE of the optimized formula with drastic improvement in the stability of SP micellar systems by mixing with F127 and P123. SP-MPM also showed a drastic increase in the release profile his improvement in the drug release can be attributed to enhanced solubilization of MN in the SP-MPM micellar systems owing to the increased hydrophilicity. In addition, These results are attributed to the presence of MN in the amorphous form within the nano-micelles confirmed by DSC and XRD measurements.

In vivo evaluation of the prepared SP-MPM was done to ensure the ability of the formula to overcome the natural clearance mechanisms of the eye and to deliver the drug effectively. This is manifested in the susceptibility test to evaluate the level of MN in the external eye tissue of albino rabbits following the topical application of SP-MPM compared to MN suspension. The results showed a higher percentage inhibition of bacterial growth compared to MN suspension. Moreover, treatment with SP-MPM mixed micellar formula also showed higher therapeutic activity as well as reducing the number of viable CFU recovered thereby reducing the signs of infection. These results can be attributed to the enhancement of MN release from SP-MPM compared to free MN which in turn increased its concentration in the precorneal tear film. Moreover, The nanometric size of the formulations facilitated their passage through the hydrated network of the corneal stroma with the non-ionic surfactants loosening the tight junctions of the corneal epithelial barriers, allowing the penetration of MN via the paracellular route ⁴⁸. Simultaneously, the ability of SP-MPM micellar systems to form in-situ gelling in the eye aided in retaining the formulation in the eye and guarded against its loss due to natural clearance mechanisms of the eye such as blinking and tears⁴⁷. Histopathological examination of both treatment groups, as well as positive and negative controls, confirmed the efficacy of the micellar formulation after 7 days of treatment in rabbit’s eyes with marked attenuation of inflammation and the return of the eye to the normal conditions compared to MN suspension due to MN hydrophobicity and inability to permeate the ocular tissue and consequently it is unable to reach and treat deep eye tissues such as the retina which favor the passage of hydrophilic molecules⁴⁹. The results also demonstrate the safety of the prepared formula and the absence of any irritant or degenerative effect of the individual components of the prepared formulas either MN or the polymers on the eye tissue⁵⁰.

CONCLUSION:

Based on the work in this research, it can be concluded that SP-pluronic polymeric nano-micelles offer a successful ocular delivery platform for broad-spectrum antifungal drug MN. The prepared nano-micelles had particle sizes lower than 100nm as well as high encapsulation, adequate drug release, and stability. The prepared micellar system also showed successfully in vivo capabilities compared to MN suspension in the treatment of ocular candidiasis with high penetration and treatment with the absence of any irritation ensuring both the safety and efficacy of the prepared micellar formula compared to a standard drug suspension.

ACKNOWLEDGMENT:

We would like to thank Mahmoud Galal, Mohamed Alteelab, Sara Mohamed and Hadeer Taha for their assistance throughout various aspects of our study.

CONFLICT OF INTEREST:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Received on 03.05.2021 Modified on 01.08.2021

Research J. Pharm.and Tech 2022; 15(2):501-511.

DOI: 10.52711/0974-360X.2022.00081

Formulation	Polymer (%w/v)			%EE	Particle size (nm)	PDI	CP(°C)	%Q₆
Formulation	SP	P123	F127	%EE	Particle size (nm)	PDI	CP(°C)	%Q₆
SP- MN	1.5	-	-	28.13±3.68	55.73 ± 4.03	0.195	35.58±2.79	64.04±2.15
SP-MPM	1.5	5	5	99.19±7.03	44.39 ± 2.68	0.138	76.95± 2.12	83.48±3.79