Formulation and Evaluation of Surfactants Based Ocular Drug Delivery System of Antifungal Drug

The structure of eye is divided into two main parts that are anterior segment and posterior segment. Approximately one-third portion of the eye is occupied by anterior segment whereas the residual portion is occupied by the posterior segment. Anterior portion includes cornea, aqueous humor, conjunctiva, iris, ciliary body and lens whereas back of the eye or posterior segment of the eye consists of sclera, choroid, neural retina, retinal pigment epithelium, optic nerve and vitreous humour. ^[1,2]

Ideal characteristics required to optimize ocular drug delivery systems:

1. It should have good corneal penetration.
2. It should possess prolonged contact time of drug with corneal tissue.
3. There should be simplicity in instillation and removal for the patient.
4. It should be non-irritating to eye.

Routes of Ocular Drug Delivery

1. 1. Topical route
   2. Peri ocular route

It includes subconjunctival, subtenon, retrobulbar, and peribulbar administration

1. 3. intravitreal route

VESICULAR DRUG DELIVERY SYSTEM

In novel drug delivery system, the vesicular drug delivery system (VDDS) has become popular due to its benefits such as improved bioavailability and reduced dose frequency. It gives prolonged  and controlled action at the corneal surface by preventing the metabolism of the drug from the enzymes present at the tear/corneal epithelium

PREPARATION OF SURFACTANT BASED VESICLES

They can be prepared by theethanol injection method.

Dissolve drugin ethanol in water bath at 70 ˚C

↓

Quickly inject this resultant solution into preheated aqueous phase containing the EA(Tween80)

↓

Stir continuously on a magnetic stirrer at 1000 rpm for 15 min at 70 ˚C

↓

Cool at room temperature to get drug-loaded surfactant based vesicular dispersion^[14]

• • Uveitis
  • Diabetic retinopathy

FUNGAL INFECTIONS

Fungal infections are mostly responsible for ocular morbidity and blindness. Fungi are subdivided into yeasts and molds. For treatment of ocular fungal infection antifungal agents are required. ^[16]

MATERIALS AND METHODS

Materials:-

1. Drug: Voriconazole
2. Chemicals: Chemicals and reagents used for the preparation of  buffers, solutions and other experimental purposes are listed in table.

List of chemicals used in formulation of eye drops

All the other reagent sused were of analytical gradeand were used as procured.

3. Instruments: Instruments and equipments used.

List of Equipment’s used

Method:-

Preformulation studies

Characterization of drug

1. Description: The sample was analyzed for its nature, colour and odour.
2. Melting Point: Melting point of Voriconazole was determined by micro-controlled based melting point apparatus (Chemiline-CL 726). Taking a pinch of  Voriconazole into a capillary tube, closed at one end. Then the capillary was inserted in bath of silicone oil which was heated in controlled manner with the help of electrical heating coil. The temperature which made drug sample transparent was noted as melting point temperature. Average of triplicate readings was noted and compared with the literature value. ^[26]
3. Differential Scanning Calorimetry (DSC): DSC was performed in order to assess the thermal behavior of the drug. It measures heat flow in and out of both sample and reference during control temperature. About 1 mg of the sample was sealed in the aluminium pan and heated at the rate of 10°C/min, covering a temperature range of 30°C to 35°C under nitrogen atmosphere, at flow rate of 20 ml/min. ^[27]
4. FTIR Spectroscopy: IR study was carried out to check purity of drug. It was determined by Fourier Transform Infrared spectrophotometer. The IR spectrum of Voriconazole was recorded using Fourier transform infrared spectroscopy (FTIR) to check its purity. The spectrum was recorded over the wave number of 4000 to 400 cm-1. ^[28]
5. UV spectroscopy:
   • λ max determination ^[29]

Accurately weighed 100 mg of drug was dissolved in 100 ml of ethanol to form stock-I (1000μg/ml).10 ml ofsolution was with drawn from stock-I added to 100 ml of volumetric flask. The volume was made to 100 ml using ethanol to form stock-II (100 μg /ml). Then 1 ml from stock-II was withdrawn and added to 10 ml volumetric flask. The volume was adjusted to 10ml using ethanol to prepare final solutions (10μg/ml). The UV spectrum was recorded in range of 200-400 nm using UV spectrophotometer. The ƛ max was determined by scanning solution of 10μg/ml against blank solution..

• • Calibration Curve of Voriconazole ^[29]
• Preparation of Primary stock solution:

Accurately weighed 100mg of voriconazole was added in to 100 ml volumetric flasks. Then small amount of ethanol was added to dissolve the drug and then volume was made to 100 ml with the methanol (diluents). The concentration of standard stock solution was 1000 µg/ml.

• Preparation of Secondary stock solution:

Transferred 1 ml from the above standard stock solutions in to 10 ml volumetric flasks and diluted up to the mark with diluent toget working secondary stock solution having concentration of 100μg/ml.

• Preparation of diluted concentration:

From the secondary stock solution 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4 ml were transferred to 10ml volumetric flasks and final volume was made to 10ml with methanol to prepared solution in concentration range of 10-24 μg/ml. The absorbance of each dilutions were measured at λ max 255 nm using methanol as ablankand standard curve was plotted between concentration (μg/ml) on X-axis and absorbance on Y-axis. The calibration curve follows equation of straight line,

Y = mx + C

Where, Y=absorbance  m=slope  x=concentration  C=constant

6. Drug Excipient Interaction Study: Drug- excipient interaction study was performed by FTIR and  DSC studies. IR was determined by Fourier Transform Infrared Spectrophotometer (FTIR-410,Jasco, Japan). The spectra were scanned over wavelength region of 4000 to 400 cm-1 at resolution of 4 cm-1. ^[28] For DSC study, about 1mg of the sample was sealed in the aluminum pan and heated at the rate of 10˚C/min, covering a temperature range of 30˚C to200˚C under nitrogen atmosphere, at flow rate of 20 ml/min. ^[27]

Characterization of excipients

1. Saponification value:

Saponification value is number of mg of potassium hydroxide necessary to neutralize the free acids and to saponify esters present in 1gm of substance. 2 gm. of substance under examination was accurately weighed into 200ml flask of borosilicate glass fitted with reflux condenser. Then in this flask 25ml of 0.5M ethanolic KOH and little pumice powder was added.All this material was boiled under reflux on water bath for 30min. 1ml of phenolphthalein solution was added and this whole solution was titrated with 0.5M HCl (a). Blank titration was carried out (b). Saponification value was determined by using formula-

Saponification Value=28.05×(𝑏−𝑎)/𝑊

Where, W- Weight in gm of substance [30]

2. Acid value:

Acid value is the number which express in milligrams. The amount of potassium hydroxide necessary to neutralize the free acids present in 1 gm of the substance. 10 g of the substance under examination was accurately weighed into 50ml flask of a mixture of equal volume of ethanol (95 %) and ether previously neutralized with 0.1 m potassium hydroxide to phenolphthalein solution. If the sample does not dissolve in the cold solvent connect flask with the reflux condenser and warm slowly with frequent shaking until the sample dissolve. Add 1 ml of phenolphthalein solution and titrate with 0.1 KOH until the solution remains faintly pink after shaking for 30 seconds. Acid value was determined by using formula-

Acid value=5.61 × n/𝑊

Where, n= the number of ml of 0.1 M KOH required. W=the weighting of the substance[30]

Preparation of surfactant based vesicles:

Surfactant based vesicles containing Span 80 and edge activator (EA) Tween 80 were prepared by ethanol injection method. Voriconazole and span 80 were dissolved in ethanol. The solution so prepared is then transferred rapidly into the preheated tween 80 and vigorously stirred on the magnetic stirrer at high speed 1000 rpm/ min. Voriconazole was used at a concentration of 10 mg/ml for the preparation of vesicles. [12,14]

Ethanol injection method for formulation of Surfactants Based Ocular Drug Delivery system of voriconazole.

Franz diffusion cell

DSC thermogram of pure drug

FTIR Spectrum of  Voriconazole

UV Spectrum of Voriconazole

Sr.no	Concentration (µg/ml)	Absorbance
1	0	0
2	10	0.2898
3	12	0.3543
4	14	0.4321
5	16	0.4982
6	18	0.5687
7	20	0.6254
8	22	0.6532
9	24	0.7132

Calibration Curve of Voriconazole in ethanol

FTIR spectra of Surfactants Based Ocular Drug Delivery system of Voriconazole

DSC thermogram of Optimized formulation

Characterization of excipients (span80 and tween80)

1. Saponification value-

Saponification value was determined by using formula-

Saponification value=28.5  x (b-a)w

 

Saponification value span 80 =28.5  x (20.5-10.5)2

 

=140.25

Saponification value of span 80 was found near to standard value (145-160) and it confirmed the purity of span 80.

Saponification value Tween 80 =28.5  x (20.5-16.2)2

 

=60.30

Saponification value of tween 80 was found near to standard value (45-55) and it confirmed the purity of tween 80.

2. Acid value-

Acid value was determined by using formula-

Acid value =5.61  nw

 

Where, n= the number of ml of 0.1 M KOH required.

W= the weighting of the substance.

Acid value for Span 80 =

Acid value =5.61  15.610

 

=8.75

Acid value of span 80 was found near to standard value (less than 8) and it confirmed the purity of span 80.

 Acid value of tween 80 =

Acid value =5.61  2.510

 

=1.40

Acid value of tween 80 was found tobe complied with standard value and it confirmed the purity of tween 80.

Evaluation of surfactant-based formulation

1. Drug content

Drug content of all the batches of formulation was calculated. Drug content was calculated by using formula

%Drug content =actual drug content/The oretical drug content X 100

The result was found in the range of 73.72-80.03%. The drug content of the F4 formulation was found to be higher i.e., 80.03% based on the calibration curve. All the values of formulation are shown in table no 9.6.

Drug content of all batches of formulation

Sr. No	Formulation Batch	Drug content (%) ± SD (n= 3)
1	F1	76.26±0.0124
2	F2	76.41±0.0152
3	F3	74.11±0.0100
4	F4	80.03±0.0152
5	F5	77.42±0.0100
6	F6	77.68±0.0152
7	F7	76.21±0.0200
8	F8	78.10±0.0152
9	F9	73.72±0.0200

Response 3D Surface plot for Drug Content(%)

Comparative drug diffusion profile of F1- F9

Response 3D Surface plot for CADD (%)

Sr. No	Time (min)	CADD/cm2 ± SD (n=3)	CADD % ± SD (n= 3)
1	0	0	0
2	30	0.03±0.01	0.70±0.01
3	60	0.04±0.01	1.00.±0.01
4	90	0.2±0.05	4.56±0.11
5	120	0.46±0.01	10.50±0.1
6	150	1.43±0.05	32.30±0.10
7	180	1.46±0.12	33.30±0.27

CADD% in saline pH 7.4 solution

Optical microscopy of optimized formulation batch 4

Graph of particle size of optimized batch (F4)

Graph of zeta potential of optimized batch (F4)

Extent of drug aggregation in refrigerator and at ambient room temperature (25˚C) was significantly low. Hence, the formulation can be refrigerated or stored at room temperature for use.

CONCLUSION

Surfactant based vesicles of  Voriconazole containing Span 80 and edge activator (EA) Tween 80 were prepared by ethanol injection method and evaluated.

Purity of drug and excipients were confirmed by pre-formulation testing. Purity of drug was confirmed from calibration curve, melting point and analytical method. IR and DSC studies indicated excipients used in formulation were compatible with drug.

By using 3² full factorial design, surfactant-based vesicles of Voriconazole were prepared in which concentration of tween 80 and concentration span 80 were used as 2 factors and experiments were performed. The response surface plot was generated using software. This was used to observe the effect of independent variables on the % drug content and corneal permeability. From plot it was observed that formulation batch F4 containing 20:80 (Span 80: Tween 80) showed better corneal permeability (95.83±0.04 %) performed on goat’s eye and higher drug content (80.03±0.0152 %) among all formulated batches.

When optimized batch is compared with normal saline pH 7.4 solution it found that newly developed surfactant-based formulation gives increase in corneal permeability.

In stability study,it was found that extent of drug aggregation upon storage in refrigerator and at ambient room temperature was significantly low. Hence the formulation is stable in nature.

Therefore, from the present study it can be concluded that surfactant based ocular drug delivery systems shows good permeability for topical ocular delivery of Voriconazole and it can be used to deliver drugs to the posterior segment of the eye.

REFERENCES

1. Kumar, S., & Bhowmik, D. (2012). Ocular inserts: A novel controlled drug delivery system. The Pharma Innovation Journal, 1(1), 1–16.
2. Patel, A., Cholkar, K., Agrahari, V., & Mitra, A. K. (2013). Ocular drug delivery systems: An overview. World Journal of Pharmacy, 2, 47–64.
3. Kotecha, R., & Mangi, R. (2013). Advances in ophthalmic drug delivery system. International Journal of Pharmaceutical Sciences, 4, 17–31.
4. Modi, K., & Shelat, P. (2012). Applications of novel vesicular drug delivery system as ocular drug vehicles: A review. International Journal of Pharmaceutical Sciences and Research, 3, 4554–4561.
5. Sharma, U., Verma, A., Prajapati, S., & Pandey, H. (2013). Ocular drug delivery: Assorted obstructions and contemporary progresses. International Journal of Research in Drug and Pharmaceutical Sciences, 2, 464–473.
6. Kumar, M., & Kulkarni, G. (2011). Recent advances in ophthalmic drug delivery system. International Journal of Pharmacy and Pharmaceutical Sciences, 4, 387–394.
7. Tangri, P., & Khurana, S. (2011). Basics of ocular drug delivery systems. International Journal of Research in Pharmaceutical and Biomedical Sciences, 2, 1541–1552.
8. Rathore, K., & Nema, R. (2009). An insight into ophthalmic drug delivery systems. International Journal of Pharmaceutical Sciences and Drug Research, 1(1), 1–5.
9. Malhotra, M., & Majumdar, D. K. (2001). Permeation through cornea. Indian Journal of Experimental Biology, 39, 11–24.
10. Kumavat, S., Chaudhari, Y., & Borale, P. (2013). Transfersomes: A promising approach for transdermal drug delivery system. Asian Journal of Pharmaceutical Science and Research, 3, 1–17.
11. Patel, C., & Majumdar, S. (2014). Patent review on surfactant-based elastic vesicles for ophthalmic drug delivery system. Inventi Journals, 1–9.
12. Kakkar, S., & Kaur, I. (2011). Spanlastics: A novel nanovesicular carrier system for ocular delivery. International Journal of Pharmaceutics, 202–210.
13. Sahoo, R., Biswas, N., & Guha, A. (2014). Nonionic surfactant vesicles in ocular delivery: Innovative approaches and perspectives. BioMed Research International, 2014, 1–13.
14. Basha, M. (2013). Design and optimization of surfactant-based nanovesicles for ocular delivery of clotrimazole. Journal of Liposome Research, 202–210.
15. Jiao, J. (2008). Polyoxyethylated nonionic surfactants and their applications in topical ocular drug delivery. Advanced Drug Delivery Reviews, 60, 1663–1673.
16. Williams, R., & Taft, D. (2008). Advanced drug formulation design to optimize therapeutic outcomes. In Ophthalmic infections and therapeutic strategies (pp. 143–163).
17. Kernt, M., & Kampik, A. (2010). Endophthalmitis: Pathogenesis, clinical presentation, management and perspectives. Clinical Ophthalmology, 4, 121–135.
18. DrugBank. (2015, November 12). Voriconazole. Retrieved from https://go.drugbank.com/drugs/DB00582
19. Rowe, R. C., Sheskey, P. J., & Quinn, M. E. (2009). Handbook of pharmaceutical excipients (6th ed.). Pharmaceutical Press.
20. Sharma, A., Pathak, M., & Sharma, D. (2012). Recent treatment strategies of posterior segment diseases of eye: A review. Novel Science International Journal of Pharmaceutical Science, 5, 254–258.
21. Chauhan, M., Sharma, D., & Arora, S. (2015). Carrier centric approach of targeting drugs to the posterior segment of the eye. International Journal of Therapeutic Applications, 22, 20–35.
22. Patel, V., & Agrawal, Y. (2011). Current status and advanced approaches in ocular drug delivery system. Journal of Global Trends in Pharmaceutical Sciences, 2, 131–148.
23. Sasaki, H., Karasawa, K., & colleagues. (2013). Retinal drug delivery using eyedrop preparations of poly-L-lysine-modified liposomes. European Journal of Pharmaceutics and Biopharmaceutics, 83, 364–369.
24. Vodithala, S., Khatry, S., & Shastri, N. (2010). Development and evaluation of thermoreversible ocular gels of ketorolac tromethamine. International Journal of Biotechnology, 1, 39–45.
25. Malhotra, S., & Khare, A. (2014). Design and evaluation of voriconazole eye drops for the treatment of fungal keratitis. Journal of Pharmaceutics, 2014, 1–6.
26. Scott Smith Online. (2016). Measurement of melting point. Retrieved July 16, 2016, from http://www.scottsmithonline.com/interests/medicalschool/chemistry/104a/file_storage/CourseWebSite/www.vanderbilt.edu/AnS/Chemistry/general_chem/104atest/measurement_of_melting_point.htm
27. Kanse, S. (2014). Solubility and dissolution rate enhancement of antifungal voriconazole by hot melt extrusion and development of sustained release tablets. World Journal of Pharmaceutical Research, 3, 1827–1853.
28. Pavia, D. L., Lampman, G. M., & Vyvyan, J. R. (2007). Introduction to spectroscopy (4th ed.). Cengage Learning.
29. Shaik, R. (2012). Development and validation of new analytical method for voriconazole for injection by using UV spectrophotometer. International Journal of Advances in Pharmaceutical Sciences and Biomedical Management Sciences, 240–247.
30. Indian Pharmacopoeia Commission. (2007). Indian Pharmacopoeia (Vol. 1, pp. 80, 89). Government of India, Ministry of Health and Family Welfare.
31. Amrutkar, C. S., et al. (2023). Recent advances in nanocarrier-mediated ocular drug delivery systems. Pharmaceutics, 15(8), Article 1862. https://doi.org/10.3390/pharmaceutics15081862
32. Luhar, M., et al. (2025). Polymeric micelles for ocular drug delivery: Advances and future perspectives. Journal of Ocular Pharmacology and Therapeutics. Advance online publication. https://doi.org/10.1089/jop.2024.0060
33. Li, S., et al. (2023). Nanotechnology-based ocular drug delivery systems: Overcoming ocular barriers and improving therapeutic efficacy. Journal of Nanobiotechnology, 21, Article 192. https://doi.org/10.1186/s12951-023-01992-2