The optimized cubosomal dispersion exhibited spherical nanosized particles and reasonable EE% along with higher FCZ corneal permeation (twofold) as compared to that of FCZ solution. Moreover, the in vivo study proved the efficacy and safety FCZ-loaded cubosomal dispersion in treatment of induced keratomycosis in rats compared to aqueous FCZ solution after topical ocular application. Based on the previous results, the use of cubosomal dispersion as an ocular drug delivery system is expected to improve antifungal activity of FCZ in treatment of fungal keratitis.

1. In vitro and in vivo evaluation of cubosomal nanoparticles as an
ocular delivery system for fluconazole in treatment of
keratomycosis
DEPARTMENT OF PHARMACEUTICAL SCIENCES;
GURU JAMBHESHWAR UNIVERSITY OF SCIENCE & TECHNOLOGY
(HISAR-125001)
Presented by:
Sidharth Mehta
Regn. No. 190121220005
M-pharm. (Pharmaceutics)
Session 2019-2021
Presented to:
Dr. Munish Ahuja
Professor
Department of Pharmaceutical Sciences
GJUS&T; Hisar

2. Contents
5. Characterization
a. Particle size analysis
b. Entrapment efficiency %
c. Morphology of FCZ-loaded
cubosomal nanoparticles
d. Ex vivo corneal permeation study
e. In vivo studies
6. Results and
discussion
a. Analysis of parameters
2. Introduction
a. About disease (Keratomycosis)
b. Drug profile (Fluconazole)
c. Delivery system (Cubosomes)
3. Materials and methods
a. Materials
b. Experimental design
4. Preparation
a. Emulsification method
1. Abstract
a. Keywords
7. Conclusion

3. Abstract
Objective- enhance ocular antifungal activity of fluconazole (FCZ) in treatment of keratomycosis through
incorporation into cubosomal nanoparticles.
Method- prepared by emulsification method according to 2³ full factorial design. Design-Expert® software was used to
study the effects of different formulation factors on properties of FCZ-loaded cubosomal dispersions and select the
optimal formulation. Eight FCZ-loaded cubosomal dispersions were prepared and were in vitro and in vivo evaluated.
Evaluation- In vitro, the results revealed that the optimum formula exhibited a mean particle size of 48.17 ± 0.65 nm
and entrapped 85.70 ± 2.56% of FCZ. The ex vivo permeation study confirmed a two-fold enhancement in FCZ
permeation through rabbit cornea compared to aqueous FCZ solution. Furthermore, in vivo ocular tolerance and
histopathological studies proved the efficacy and safety FCZ-loaded cubosomal dispersion in treatment of induced
keratomycosis in rats compared to aqueous FCZ solution after topical ocular application.
Conclusion- The obtained results indicated that cubosomes could be promising ocular drug delivery system for
enhancing antifungal activity of FCZ in treatment of fungal keratitis in rats.
Keywords- Fluconazole, Cubosomes, Corneal permeation, Ocular delivery, Keratomycosis

4. Introduction
a. About disease (Keratomycosis)
Normal human eye
Keratomycosis infected eye
• Fungal keratitis (keratomycosis) is a serious disease that can lead to
loss of vision.
• Its incidence rate is increasing due to widespread use of contact
lenses, especially bandage contact lenses, and topical steroid usage.
• Causing organism- Candida species.
• Management of fungal keratitis remains a challenge because of the
protective mechanisms of the eye and the poor corneal penetration of
antifungal drugs.

5. b. Drug profile (Fluconazole)
Description- Fluconazole (FCZ) is a hydrophilic bis-triazole compound having broad spectrum antifungal activity.
Molecular formula- C13H12F2N6O
Molecular weight- 306.271 g·mol−1
Therapeutic uses- 1. Candidiasis
2. blastomycosis
3. coccidiodomycosis
4. cryptococcosis
5. histoplasmosis
6. dermatophytosis
7. pityriasis versicolor

6. Problems associated with delivery of fluconazole-
• Solutions and suspensions of antifungal drugs formulated as eye drops are available for topical
ocular administration.
• However, their active constituent becomes diluted in tear film as the preparation is instilled into
the cul-de-sac and is rapidly drained away from pre-corneal cavity by constant tear flow
and lacrimo-nasal drainage that lead to low drug ocular bioavailability, which is the most challenging
in ocular formulation.
Solution to above problem-
• Several formulation approaches have been attempted to improve FCZ ocular.
• The objective of this research was to investigate the potential of cubosomal nanoparticles as an
ocular delivery for FCZ that would allow greater permeation, longer contact time, improve
bioavailability, and reduce ocular irritancy of the drug in treatment of fungal keratitis.

7. c. Cubosomes
• Cubosomes are liquid crystalline phases comprising lipid bilayer enclosing water channels.
• The structure of cubosomes enables the encapsulation of both hydrophilic and hydrophobic drugs
with varying solubility.
• Because of the structural similarity of cubosomal lipid
bilayer to the biological membrane, cubosomes may enhance
the drug delivery topically.
• Also, cubosomes can be easily formulated and
administered in a liquid form and seemed to have high diffusivity
across the corneal epithelium

8. Materials and methods
a. Materials
1. Fluconazole (Drug)
2. Myverol® 18–99 K (Emulsifier)
3. Poloxamer 407 (Surfactant)
4. Acetonitrile, methanol, and chloroform (Solvents)
5. Pathogenic Candida albicans wild-type (SC5314 strain)
6. Other Chemicals were of analytical grade
Fluconazole Myverol 18-99K Poloxamer 407

9. b. Experimental design
A full 2³ factorial design was used to statistically optimize the variables for the
preparation of FCZ-loaded cubosomes using Design-Expert® version 7.0.0
software (Estat-Ease, Inc., Minneapolis, Minnesota, USA)

10. Preparation
FCZ-loaded cubosomal dispersions were prepared by emulsification method.
GMO (Myverol ® 18–99 K) Poloxamer 407
Molten in a 70°C water bath
Fluconazole Ethanol (5 ml)
Dispersed
Heat the obtained solution at 40°C to evaporate ethanol
Obtained mixture was dropped into 4 ml. of deionized water preheated at 70°C under mechanical stirrer
Prepared cubosomal dispersions are sonicated either for 2 or 4 min and then stored in amber glass vials

11. Characterization
a. Particle size analysis
• The particle size distribution and polydispersity index (PDI) of cubosomal nanoparticles were
determined using Zeta Sizer Nano-series (Nano ZS), Malvern, Worcestershire, UK.
• Samples were suitably diluted and measured at 25 ± 0.5 °C in triplicate.
b. Entrapment efficiency %
• FCZ-loaded cubosomal dispersion (1 mL) was suitably diluted with deionized water and centrifuged
at 6000 rpm for 15 min.
• Free FCZ contained in filtrate was measured using a validated HPLC assay.
%EE= [Entrapped amount of FCZ/ total amount of FCZ] × 100
c. Morphology of FCZ-loaded cubosomal nanoparticles
Performed using a transmission electron microscope. A droplet of cubosomal dispersion was adsorbed on
carbon-coated copper grid, and the excess fluid was removed by air drying at room temperature. The
samples were negatively stained with 1% sodium phosphotungstate solution before TEM examination.

12. d. Ex vivo corneal permeation study
• Rabbits corneas used in this study were freshly isolated and immersed in simulated lacrimal fluid (pH 7.4) for 30 min
prior to the experiment.
• Then cornea used was fixed in position to top-cut plastic syringe representing a dialyzing tube providing an effective
permeation area of approximately 0.739 cm² .
• The cornea was placed with its outer epithelium layer facing upward (inside of the tube) and innermost endothelium
layer facing the permeation medium.
• The tested preparations (F4 and FCZ aqueous solution, 0.2% w/w) were accurately placed in the plastic tube on the
isolated cornea at an amount equivalent to 0.5 mg of drug.
• A 250 μL aliquot of the permeation medium was withdrawn at time intervals (1, 2, 3, 4, and 5 h) and was replaced
with an equal volume of fresh medium to maintain a constant volume. All samples—in triplicates—were filtered through
a 0.22-μm pore size syringe filter and analyzed by HPLC.

13. e. In-vivo studies
1. Sterilization cubosomal dispersion (F4) and FCZ aqueous solution-
By autoclave at 121ºC for 15 mins.
2. Experimental animals-
24 adult male Wistar rats weighing 230–250 g were used.
3. Induction of keratomycosis
• For rapid induction of infection, rats were immunocompromised with methyl prednisolone (50 mg/kg) for
3 consecutive days.
• After anesthetization with intraperitoneal ketamine (50 mg/kg)-xylazine (10 mg/kg) mixture,the right eye cornea of
each immunocompromised rat was scarred using a 27-gauge needle followed by the application and even distribution
of a 5 μl suspension containing Candida albicans wild-type (SC5314 strain).
• A clear visual cloudy appearance of corneas was observed on the fourth day after Candida albicans inoculation.
4. Experimental design- Immunocompromised rats were divided into 4 groups (6 rats/group) treated as follows:
(a) normal group (untreated rats)
(b) CC group (rats were inoculated with Candida albicans without any treatment)
(c) CC + FCZ aqueous solution group (rats inoculated with Candida albicans and treated with FCZ aqueous solution)
(d) CC + FCZ loaded cubosomal dispersion group (rats were inoculated with Candida albicans and treated with FCZ-
loaded cubosomal dispersion). Dose- 50 μl

14. 5. Histological examination
• At the last day of experimental protocol, animals were euthanized by cervical dislocation. Autopsy
samples were taken from the eye of rats of different groups, and fixed in 10% formalin for 24 h.
• The obtained tissue sections were stained by hematoxylin & eosin stain and examined by the light electric
microscope.

15. Results and discussion
a. Analysis of factorial design
The effects of three formulation variables (X, X2, and X3) each at 2 levels on (Y1), particle size (Y2), and zeta
potential (Y3) were statistically evaluated using ANOVA (partial sum of squares–type III) at P < 0.05. The
design was evaluated using following polynomial equation:
Y= β0 + β1X1 + β2X2 + β3X3 + β4X1X2 + β5X1X3 + β6X2X3
where,
Y is the response variable,
β0 is constant and
β1, β2, β3, β4, β5, and β6 are the regression coefficients.
X1, X2, and X3 stand for the main effect
X1X2, X1X3, and X2X3 are the interaction terms

18. b. Particle size of the prepared FCZ-loaded cubosomes
The mean particle size for the prepared FCZ-loaded
cubosomes ranged between 40.53 ± 0.54 and 116.10
± 1.35 nm

19. c. EE% of the prepared FCZ-loaded cubosomes
d. Zeta potential of the prepared FCZ-loaded cubosomes
The EE % for the prepared FCZ-loaded cubosomes
ranged between 47.29 ± 1.37 and 85.70 ± 2.56%
The values of zeta potential of the prepared FCZ-loaded
cubosomes ranged between − 21.30 ± 0.22 to − 35.80 ±
0.56 mV

20. e. Morphology of FCZ-loaded cubosomal nanoparticles
TEM photograph of the selected cubosomal formulation (F4) reveals that the FCZ-loaded cubosomal
nanoparticles are spherical in shape without aggregation.

21. f. Ex vivo corneal permeation
• Permeation of FCZ from the selected cubosomal formula (F4) through rabbit cornea in comparison with aqueous
FCZ solution. The results illustrated in Fig. 4 showed that cubosomes F4 provided approximately twofold enhancement
in the drug permeation through rabbit cornea.
• The flux of FCZ obtained from cubosomes F4 and FCZ solution was 66.74 ± 8.09 μg cm-² h-¹ and
32.07 ± 1.78 μg cm-² h-¹ respectively.

22. g. In vivo studies
1) Effect of different treatments on area % of corneal opacity
• The red color in the Imagej-processed photographs designates the
areas of focal lesions.
• The area % of corneal opacity was calculated as an indicator of
clinical presentation of Candida infection.

23. 2) Histopathological findings
• Normal group panel, there were no
histopathological alterations in the eyelid, and the normal histological
structure of the collagen and epithelial tissue was recorded.
• CC group panel, massive inflammatory cells aggregation and
edema were detected in the stromal connective tissue of the eyelid.
Also, the covering epithelium of the cornea showed more focal
stratification which is extended deep into the stroma.
• CC + FCZ aqueous solution treated panel, few inflammatory
cell infiltration and edema were detected in the connective tissue.
• There were no histopathological alterations in the iris, sclera,
choroid, or retina. However, there were no histopathological
alterations in all tissue sections from rats treated with
FCZ-loaded cubosomal dispersion.

24. 3) Histological score of inflammation
• The total inflammation was scored for different eye
tissues as follows:-
(0, no inflammation; 1, mild inflammation;
2, moderate inflammation; 3, massive inflammation).
• The total final score for each animal has been ranged from
0 to 12.
• A zero score was observed for normal animals,
• while an extremely high score was calculated for animals
of the untreated CC group.
• On the other hand, FCZ solution and FCZ-loaded cubosomal
dispersion have significantly shown a decreased
inflammation score.

25. Conclusion
The optimized cubosomal dispersion exhibited spherical nanosized particles and reasonable EE% along with higher
FCZ corneal permeation (twofold) as compared to that of FCZ solution.
Moreover, the in vivo study proved the efficacy and safety FCZ-loaded
cubosomal dispersion in treatment of induced keratomycosis
in rats compared to aqueous FCZ solution after topical ocular
application.
Based on the previous results, the use of
cubosomal dispersion as an ocular drug delivery system is
expected to improve antifungal activity of FCZ in treatment
of fungal keratitis.

26. Reference