Ophthalmic formulations are sterile in dosage, free of foreign material, and appropriately designed in a form that can be administered to the inner side (intraocular) or the cavity of the eye, or they can be used in conjunction with an ophthalmic treatment device.¹ The eye is a special and vital organ. Since the ocular route is equivalent to other routes and has a particular anatomy that can prevent drug absorption into the deep tissues of the eye, drug delivery by this channel is a very delicate task for researchers. There are many different drug delivery methods for ophthalmic usage, including eye drops, ointments, lotions and suspensions for the eyes, however, these preparations and formulations must be used with caution. Due to their solubility in tears, previously made ophthalmic dose have the potential to lose their viscosity and resistance time in the eye. This issue can be resolved by creating a medication formulation that instantly forms in situ gel when injected into the eye cavity. In situ gels were created to extend the medication’s stay in the eye cavity, improve drug resistance and bioavailability and relieve the eye from repeated doses.^2,3 The delivery methods must preserve certain properties, such as sterility, isotonicity and improved penetration from the eye muscle, in order to control the drug’s use in the ocular cavity. Less soluble in tears, less protein binding a simple installation and uninstall process, good rheological characteristics and formulation resistance in the ocular cavity^4–6 Mainly The medication is absorbed into the ocular system through the cornea, which is more efficiently than other routes like scleral and Maidenhead pericardium absorption, through which the blood vessel can absorb the drug from the absorption route and finally, general solution of the drug happens in the body. To regulate the drug’s use in the ocular cavity, the delivery techniques must maintain specific qualities such as sterility, isotonicity and enhanced penetration from the eye muscle.^7–10 Less protein binding, less solubility in tears, ease of installation and removal, good rheological properties and formulation resistance in the ocular cavity. Mainly The drug is absorbed into the ocular system through the cornea, which is more effective than other routes like scleral and Maidenhead pericardium absorption. Afterwards, the drug is generally dissolved in the body.^10–11

Dorzolamide Hydrochloride was procured from Century Pharmaceutical Limited. Natural Egg Phospholipid Powder was procured from Vitae Gen Life Sciences, Nagpur. Chitosan was procured from Bangalore Fine Chemicals, Bengaluru, Karnataka.

Liposomes containing the drug were prepared by Reverse phase evaporation technique and the prepared liposomes were then dissolved in a solution containing gelling agents. Different formulations (Formulation F1-F9) of Dorzolamide liposome prepared from different polymers with varying concentration as shown in Table 1.

It is important to look into the physical qualities of the prepared formulation. Phase separation, colour, homogeneity and consistency are examined.

FTIR was performed for the conformation of the original drug, polymer and to know polymer and drug interaction. In this test, the sample was weighed and mixed with KBr and placed under a pressure of 100 kg/cm to get pallets. FTIR analysis of the drug (dorzolamide), polymers (chitosan, egg lecithin), and physical mixture of drug-polymer and pellets were recorded. IR Spectra of a) dorzolamide sample b) Egg lecithin c) Physical mixture of drug and polymer d) Chitosan shown in Figure 1.

Figure 1:
IR Spectra of a) dorzolamide sample b) Egg lecithin c) Physical mixture of drug and polymer d) Chitosan.

Preparation of standard curve of dorzolamide hydrochloride in phosphate buffer pH 7.4: The different concentration of drug Dorzolamide 2-12 μg/ml was prepared. Then absorbance of each concentration at λ_max 253nm was taken using phosphate buffer ph 7.4 as diluent and the slope, intercept, and correlation coefficient were calculated (Figure 2). Absorbance was taken in triplicate for all the concentration and calculated the standard deviation which is shown in Table 2.

Figure 2:
Standard plot of dorzolamide hydrochloride in Phosphate buffer pH 7.4.

The pH of the gel is determined using a pH meter. The procedure involved sticking the electrode tip into the gel and 2 min later, the outcome was recorded. Results of pH for different formulation (F1 to F9) mentioned in Table 3.

The viscosity of the produced formulation is measured using a Brookfield viscometer (Brookfield LV, spindle 64). The spindle of the viscometer was allowed to rotate at predetermined rates (100 rpm) while the gel sample was placed within a glass container. The viscosity results are shown in below Table 3.^12–15

All the prepared liposomal formulation were evaluated for Spreadability and checked for layer separation by the method of centrifugation. The outcomes shown in Table 3.

The morphology, size, and size distribution of liposomal vesicles were analyzed by field emission scanning electron microscopy (SEM, ZEISS) and Transmission electron microscopy (TEM).¹³¹ SEM and TEM images of formulated liposome (Formulation F5) shown in Figure 3 and Figure 7 respectively.

Figure 3:
SEM Images of formulated liposomes.

Figure 4:
Comparative chart of Time v/s swelling index of different formulation (F1 to F9).

Figure 5:
Drug containing liposomes Polydispersity Index .

Figure 6:
Zeta Potential image of prepared liposome.

Figure 7:
TEM images of prepared Liposomes.

To test the swelling of ocular gel containing Dorzolamide hydrochloride liposomal formulations as shown in Figure 8, three set of each formulation were weighed and put in a mesh basket and inserted into phosphate buffer saline (PBS) of pH 7.4 maintained at temperature 32 ± 0.5°C at different time intervals (1 hour, 2 hour, 3 hour, 4 hour and 5 hour). The swollen formulation than removed, wiped with lint free tissue to remove excess surface PBS, weighed, and then return back to the same container.

Figure 8:
Ocular Gel Containing dorzolamide Liposome.

Swelling index was calculated using the following equation:

Where W0 is the initial weight of the sample and Wt is its weight at time t. Results of swelling index for formulated liposomes (F1 to F9) summarized in Table 4 and comparative chart of time v/s swelling index of different formulation (F1 to F9) shown in Figure 4.

The size and polydispersity index of the different liposomes were obtained from DLS measurements by using a Zeta sizer nano ZS Malvern Instrument. Samples were illuminated with a laser at a fixed detection arrangement of 90° to the center of the cell area and fluctuations in the intensities of the scattered light were analysed. Polydispersity index of formulation F5 shown in Figure 5 and zeta potential of formulation F5 shown in Figure 6.

1 g of gel sample was dissolved in 100 mL of phosphate-saline buffer, pH 7.4 and then sonicated for 2 hr. Before the sonicated mixture was exposed to UV analysis, it was filtered using a 0.45 m Millipore filter.^16–18

A Franz diffusion cell is used to perform drug release studies. 200 mg of emulgel is evenly distributed across the surface of the egg membrane. Both the chambers of the diffusion cell are sandwiched together by the egg membrane. To solubilize the medication, newly made PBS solution (pH 5.5) is poured into the receptor chamber. The receptor chamber is stirred using a magnetic stirrer. The samples are taken in 1.0 mL aliquots at predetermined intervals. After the proper dilutions, samples are examined using a UV-visible Spectrophotometer to determine their drug content. Cumulative adjustments are made to establish the overall amount of medication released at each time period. The total amount of medication delivered through the egg membrane is then calculated as a function of time.^18–22 Their drug content. Cumulative adjustments are made to establish the overall amount of medication released at each time period. Results of in-vitro drug release summarized in Table 5 and cumulative drug release of dorzolamide hydrochloride from in-situ ocular gel of different formulation (Formulation F1 to F9) shown in Figure 9.

Figure 9:
% Cumulative Drug release of Dorzolamide Hydrochloride from in-situ ocular gel.

Figure 10:
Kinetic drug release study for F1to F9 formulation.

It was found that the melting point of dorzolamide was 284.4°C.

In an IR study, it was discovered that the sample’s peaks and the dorzolamide reference spectra from IP had similar peak heights. Similar wavenumber range peaks are visible in the scan as in the reference spectra. It was therefore expected that the sample would be found to be pure dorzolamide and suitable for use in further research.^22–25

Kinetic drug release study for F1 to F 9 formulation.

In this study, the potential of liposomes as a drug vehicle for the ophthalmic delivery of dorzolamide was investigated. Nine liposomal formulations have been designed and optimized. Physical characterization revealed that liposomes embedded in situ ocular gel are suitable for ocular administration, with an encapsulation efficiency of 90%. The formulations stored at +4°C were stable for two months and greater chemical stability was obtained with the HA coating. Liposomes guarantee a prolonged and gradual release and they improve the residence time of the drug into the eye and also enhance the bioavailability of the drug by enhancing the corneal penetration of the drug.

Finally, the in vitro uptake study, conducted with fluorescent liposomes, showed that liposomal formulations increased the absorption at the cellular level and, in particular, at nucleus level, when tested in the corneal and conjunctival cells, with the most marked effect for HA-coated liposomes.

The authors would like to thank whole faculty members and management of SGRRU (School of Pharmaceutical Sciences), Patel Nagar, Dehradun- 248001 for providing all necessary technical support and motivating us at every stage.