Design development and evaluation of novel ophthalmic nano lipid in situ gel-forming solution using timolol hydrochloride

Mohd Azharuddin*1, Theivendren Panner Selvam2, Maya Sharma3, Jayesh Dwivedi4 1Paci ic Academy of Higher Education and Research University, Udaipur, Rajasthan, India 2Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy, Elayampalayam, Namakkal, Tamil Nadu, India 3Department of Pharmaceutical Chemistry, Paci ic College of Pharmacy, PAHER University, Udaipur, Rajasthan, India 4Paci ic College of Pharmacy, PAHER University, Udaipur, Rajasthan, India

Timolol hydrochloride, in situ gel, entrapment ef iciency, in-vitro release, gelation study, ocular irritation ABSTRACT The present research work was aimed to design, develop and evaluate the nano lipid-based drug delivery system by incorporating timolol hydrochloride drug for ocular therapy and improve the release of the drug through the ocular route. Nanolipids in situ gels were prepared by ilm hydration method involving two steps. First nano lipids were formulated with the help of organic solvents, and then they were incorporated into a gel by using gelling agents. FTIR spectrum studies were carried out for drug and the formulations which reveal that there was no interaction between the drug and excipients used. The various formulations prepared were subjected for the different evaluation parameters, which showed good and effective results for visual appearance, pH, gelation study, viscosity and ocular irritation studies. It was further observed from this research work that formulation TF2 (HPMC K-15M 0.2%w/v and Carbopol 940 0.4%w/v) had a maximum entrapment ef iciency of 97.30%, drug content of about 97.67% and drug release of about 84.29% for 10 hrs. Stability studies were carried out for TF2 formulation, and they found that they were stable throughout the study period. It was inally concluded from the present work that formulations prepared were more suitable and had good patient compliance compared to the eye drops.

INTRODUCTION
Ocular dosage forms are being designed to be instilled onto the external surface of the eye and can be administered inside or adjacent of the eye. There has been signi icant attention to the development of controlled drug delivery systems for a few decades. The structure of the eye is unique, so it restricts the entry of drug molecules at the site of action (Gaudana et al., 2009).
Various stimuli can form gels theses includes 1. Physical stimuli: Change in temperature, electric ields, light, pressure, sound, and magnetic ields.
2. Chemical stimuli: Change in pH and ion activation from biological luids.
3. Biological or biochemical stimuli: Change in glucose level.
From the above various stimuli only pH, ion activated, and temperature stimuli can be used for designing of ophthalmic drug delivery system.
Types of in situ ophthalmic gel are 1. In situ gelling based on change in pH 2. In situ gelling based on Temperature Modulation 3. Ion-activated in situ Gellation (Nanjawade et al., 2007).
In the present research work, timolol hydrochloride nano lipid in situ gel was formulated by using the desired quantity of Carbopol 940 and HPMC K-15M (gelling agents), and other ingredients like benzalkonium chloride (preservative) and sodium chloride (isotonic with tear luid and adjust pH) were added to the gel batches in suf icient quantity.

MATERIALS AND METHODS
Pure drug of Timolol hydrochloride was purchase from Yarrow chem product, Mumbai. Carbopol 940 and HPMC K 15M were also purchased from CDH laboratory reagent, India.

Study of interaction of the drug with excipients used in the formulation
Infrared spectra of timolol hydrochloride with used lipids were recorded on FTIR spectra photometer. The absorption maxima of timolol hydrochloride in the spectrum obtained being examined in position and relative intensity to those in the IR spectra of prepared in situ gel (Eaga et al., 2009).

Formulation of nano lipids
Film hydration technique was used for the preparation of nano lipids. In round bottom lask mixture of vesicles like lecithin and cholesterol were dissolved in a volatile organic solvent (methanol). The rotary evaporator was rotated at 60ºC for 45 min. The organic solvent is removed with gentle agitation, leaving a thin ilm of lipid on the walls of a rotary evaporator.
The aqueous phase containing timolol hydrochloride drug was slowly added with intermittent, constant shaking of the lask at room temperature and followed by sonication for 30 min. Nanolipid solution was cooled by kept in 4-8ºC in the freezer.

Formulation of nano lipid in-situ gel
Nanolipid in situ gels were prepared purely based on drug entrapment ef iciency and morphology. The batch which gave maximum entrapment and good surface morphology were selected for preparation of in-situ gel. The appropriate weight of Carbopol 940 and HPMC K-15M were sprinkled over prepared nano lipid dispersion under constant stirring with the help of glass rod which avoids the formation of lumps. Benzalkonium chloride as preservative and sodium chloride to make gel formulations isotonic with tear luid were added to the gel batches in suficient quantity to adjust the pH (Table 1) (Lavanya et al., 2014).

Drug entrapment ef iciency
Ultracentrifugation method was used to separate the unentrapped drug from the nano lipid formulation where the nano lipid dispersion was centrifuged at 14000 rpm for about 90 min. The clear supernatant solution which formed was collected.
Then it's further diluted with pH 7.2 stimulated tear luid and was analyzed by UV spectrophotometric method.
Drug entrapment ef iciency was calculated by using the equation. The amount of timolol hydrochloride encapsulated in the nano lipid was determined by using EE = (Total drug concentration -free drug concentration) / total drug concentration × 100 where, EE is entrapment ef iciency (%) (Padma et al., 2010).

Estimation of drug content
50 mg of equivalent nano lipid suspension was taken into a standard volumetric lask. Then it was lysed with 100 ml of propane-1-ol by continuous shaking. Then about 0.1 ml of solution was diluted to 10 ml with stimulated tear luid 7.2. The absorbance was measured at 295 nm for timolol hydrochloride, and drug content was calculated with the help of calibration curve (Ramachandra et al., 2012;Nagesh et al., 2012).

Visual appearance and pH
Presence of particular matter can be observed by visual appearance and clarity. A digital glass electrode pH meter was used to check the pH of the formulations and pH was determined by bringing the electrode near the surface of the formulations and allowing it to equilibrate for about 1 min (Nagalakshmi et al., 2014).

In vitro gellation study
The gelling capacity of the formulations was determined by placing a drop of the polymer solution in a vial which contains about 2 ml of freshly prepared simulated tear luid (STF) pH 7.2 and equilibrated at 37ºC. Than gel formation was observed and time required for gelation and dissolution of the gel formed was noted (Moorthi et al., 2012).

Viscosity study
Brook ield viscometer was used to determine the viscosity of the formulations by using CPE -42 spindle at 10, 20, 50 &100 revolutions per min (Organisation for Economic Co-operation and Development, 2012;Sampath et al., 2012).

In vitro drug release of nano lipid in situ gel
In vitro release studies of timolol hydrochloride nano lipid, the in-situ gel was performed at 37ºC using stimulated tear luid (pH 7.2) as the release medium.
Nanolipid in-situ gel (5 ml) containing timolol hydrochloride was accurately weighed and transferred to the dialysis membrane. The gel was gently pushed down to the surface of the dialysis membrane. To wet the gel stimulated tear luid (1 ml, pH 7.2) was added in the reservoir compartment.
The receiving compartment was stirred magnetically (100 rpm) at 37ºC. At regular interval, 1 ml of samples were withdrawn from the receiving compartment, and the amount of timolol hydrochloride released from in situ gel was determined spectrophotometer at 295nm (Shimadzu1800).
After the withdrawal of the sample at a particular interval, an equal quantity of stimulated was replaced (Shashank et al., 2012).

Ocular irritation studies
Albino rabbit's six numbers were used as test species for the study. Rabbits were housed and well maintained in the animal house at room temperature (27ºC)-each weighing 2-3kg. The eyes of animals were marked as test and control.
Control animal group had not received any sample were as test group received the formulation (0.5 ml), and the animal eyes were observed for 1, 24, 48, 72 hrs. A scoring system graded ocular changes. Animals eyes were observed for redness, swelling, and watering (Nayak and Srinivasa, 2017a).

Accelerated stability studies
The optimized nano lipid dispersion, which had higher entrapment ef iciency subjected to short term accelerated stability study at 4ºC ± 2ºC and 27ºC ± 2ºC as per modi ied ICH guidelines. Samples were analyzed for drug content (Nayak and Srinivasa, 2017b).

Study of interaction of the drug with excipients used in the formulation
The FTIR spectrum studies of Timolol hydrochloride drug and in-situ gel were analyzed. Primary func-tional groups peaks {3315.52 (NH str ), 3049.90 (Ar-CH str ), 1615.26 (C=N) and 808.42 (Br)} of timolol hydrochloride were present in loaded in-situ gel prepared (Table 2, Table 3, Figure 1 and Figure 2).
This proves the fact that there was no potential interaction of the drug with the excipients used in the formulation. This indicates the stable nature of drugs in all formulations prepared.

Drug entrapment ef iciency
The entrapment ef iciency was calculated, and it is expressed in term of percentage. Entrapment ef iciency was within the range of 73.40% to 97.30% for timolol hydrochloride loaded in-situ gel formulations. TF2 formulation showed maximum entrapment ef iciency of 97.30% when compared with other formulations (Table 4 and Figure 3).

Estimation of drug content
Timolol hydrochloride loaded in situ gel formulations were determined spectrophotometrically at 295nm. Drug content of all formulations was in a range of 66.69% to 97.67%, as shown in (Table 5). The TF2 formulation showed maximum drug content of about 97.67%.

Visual appearance and pH
The presence of any particular matter was observed visually. Timolol hydrochloride nano lipid in situ gel pH range lies between 4.1-4.8 pH (Table 6). Nanolipid in-situ gel shows a maximum pH of 4.8 for TF5 formulation and minimum for TF4 with pH 4.1. The pH of the reported formulations was non-irritable to the eye.

In-vitro gellation study
Freshly prepared simulated tear luid pH 7.2 was used to determine the galling capacity. Gellation study revealed that the formulations TF1, TF3 &TF5 had a suitable gelation property and was found that gellation was maintained for about 8 hrs. Formulations TF2, TF4 & TF6 exhibited immediate gellation which remains for about 8-10 hrs as shown in (Table 7) Viscosity study The Highest viscosity for TF6 formulation leads to retarded drug release up to a considerable extent 84.29% in 10 hrs when compared with other formulations. Low viscosity formulations show the Congestion, swelling , iris reactive to light (a sluggish reaction) 1 3 Hemorrhage or no reaction to light 2 Conjuctivea 1 Normal 0 2 Some blood vessels hyperaemic (injected) 1 3 Individual vessels not easily discernible 2 4 Diffuse beefy red 3    Table 8.

In-vitro release of nano lipid in situ gel
The timolol hydrochloride nano lipid in-situ gels showed a two-step release pattern initial burst release and followed by slow release phase. An initial burst release helps to achieve the therapeutic concentration in minimal time followed by the constant release of the drug.
Developed formulations showed 35.3% drug release in 2h and 98.50% in 10h. It was observed that early release phases occurred mainly due to diffusion in the polymer matrix. In contrast, during the later phases, the release has mediated both diffusions of the therapeutic agent and degradation of the polymer matrix itself. Based on the in vitro release proiles, TF2 was selected as optimized formulations (Table 9 and Figure 4).

Ocular irritation studies
It was carried on albino rabbits. Ocular irritation studies indicate that the optimized formulation TF2 was non-irritant. It had excellent ocular tolerance, and no ocular damage or abnormal clinical signs to the cornea, iris, or conjunctivae were visible, as shown in Table 10 and Table 11.

Accelerated stability studies
The stability studies of optimized formulation TF4 timolol hydrochloride nano lipid in situ gel formulations was determined at 4 • C ± 2 • C and 27 • C ± 2 • C for 3 months. The formulation was observed visually for precipitation. The drug content was determined for every 30 days for 3 months. The drug content was determined, and there was less difference between the formulations kept at different temperatures, as shown in Table 12.

CONCLUSION
It was concluded from the above work that formulation TF2 has a maximum entrapment ef iciency of 97.30% and drug content of about 97.67%. TF2 formulation containing HPMC K-15M and Carbopol 940 about 0.2% w/v and 0.4% w/v respectively showed the drug release of about 84.29% for 10 hrs. The work concluded that the formulations prepared were more suitable and patient compliance.