Formulation and Evaluation of Brimonidine Maleate Nanolipid in Situ Gel

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


INTRODUCTION
Ideal ophthalmic drug delivery must be able to sustain the drug release and to remain in the vicinity of the front of the eye for a prolonged time. In recent years, actual observations have been concentrated on the progress of controlled and sustained drug delivery systems (CDDS& SDDS). The involved eye structure limits the access of drug at the site action (Gaudana et al., 2009;Nanjawade et al., 2007) 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.

In-situ Delivery
A new trend of preparing a in situ gel had been proposed in the year 1980s. In situ was a Latin phrase which translated literally as 'in position'.
In situ gels are low viscosity forming solutions which had been going phase change in the eye (cul-de-sac) due to the presence of polymers.
They may fallow any one of the methods like change in ionic strength or pH change or temperature etc. for phase transition on ocular systems & prolong the drug release.
The conversion of sol to gel increases the ocular time in the eye & they should not have any problem related to the vision of the eye.
Liquid eye drops when instilled into the eye, they will be drained out of the eye, which will lead to drug loss & automatically the bioavailability of the drug will reduce (1 to 10%). Thus all problems can be solved by formulation the medicine in the form of sol to gel type (insitu).
From the above various stimuli only pH, ion activated, and temperature stimuli can be used for designing of ophthalmic drug delivery system. (Nanjawade et al., 2007) In this work desired percentage of carbopol 940 and HPMC K-15M was used for the preparation of brimonidine maleate nano lipid in situ gel . (Mohan et al., 2009;Lavanya et al., 2014)

MATERIALS AND METHODS
Brimonidine maleate pure drug was purchase from yarrow chem. Product; Mumbai, India and carbopol 940 and HPMC-K-15M were purchased from CDH laboratory India.

Study of interaction of the drug with excipients used in the formula
Infrared spectra of brimonidine maleate were recorded on FTIR spectra photometer. The absorption maxima in the spectrum obtained with the substance being examined correspond in position and relative intensity to those in the spectra of in situ gel . (Preetha et al., 2010)

Preparation of nanolipids
Nanolipids were prepared by ilm hydration technique. The mixture of vesicles forming ingredients like lecithin and cholesterol are dissolved in a volatile organic in a round bottom lask. Rotate the rotary evaporator at 60ºC for 45 minutes. The organic solvent is removed with gentle agitation and evaporating the organic solvent at 60ºC and leaving a thin ilm of lipid on the wall of the rotary lash evaporator. The aqueous phase containing brimonidine maleate drugs was added slowly with intermittent shaking of the lask at room temperature followed by sonication for 30 minutes. Nanolipid solution cooled was kept in 4-8 o C at the freezer.

Formulation of nano lipid in-situ gel
The batch which provided maximum entrapment ef iciency was chosen to prepare nano lipid in situ gel. To avoid lump formation and to allow the hydration, an appropriate quantity of Carbopol 940 and HPMC K 15M have been sprinkled over nano lipid dispersion under the constant agitation with a glass rod. Benzalkonium chloride (as preservative) and sodium chloride (to make gel formulations isotonic with tear luid) were added to the gel batches in suf icient quantity (Table 1). (Ramachandra et al., 2012) Figure 1: FTIR spectra of Brimonidine maleate.

Drug entrapment ef iciency
The unentrapped drug was separated by ultracentrifugation method from the nano lipid formulation where the nano lipid dispersion was centrifuged at 14000 rpm for about 90 min. The resulting solution eliminated the transparent supernatant. The pH 7.4 phosphate buffer is diluted and analyzed according to the UV spectrophotometric methods.
Calculate the entrapment ef iciency by using the following equation. (Nagesh et al., 2012) The amount of Brimonidine Maleate encapsulated in

E E = Total drug conc -free drug conc / total drug conc x 100
Where EE is entrapment ef iciency (%)

Estimation of drug content
Nanolipid suspension equivalent to 50mg was taken into a standard volumetric lask. Then were lysed with100ml of propane-1-ol by shaking. Then 0.1ml of this solution is diluted to 10ml with phosphate buffer 7.4. The absorbance was measured at 248nm for brimonidine maleate and calculates drug content from the calibration curve. (Nagalakshmi et al., 2014;Moorthi et al., 2012)

Visual appearance and pH
For the nature of some particular matter, visual appearance and clari ication were observed. The acidic or alkaline composition of the corneal membrane is expected to cause discomfort. For this reason, there was a wireless electrode pH meter. By bringing the electrode near the surface of the formulation and allowing it to be balanced for 60sec, pH has been noted. (OECD, 2012)

In vitro gellation study
The gelling capacity was determined by placing a drop of the polymer solution in a vial containing 2 ml of freshly prepared simulated tear luid (STF) equilibrated at 37 ºC. After that, the visual assessment of the gel formation was done, and the time required for gelation and dissolution of the gel formed was noted. (Kumar et al., 2012;Nayak et al., 2012) In vitro drug release of nano lipid in situ gel A 37 • C phosphate buffer (pH 7.4) was used to test in vitro release studies for brimonidine maleate insitu gel. Brimonidine maleate containing nano lipid in situ gel (5 ml) was carefully weighed and transferred into the membrane of dialysis. Gently move the gel down to the membrane gel surface and in contact with the membrane. In the reservoir tank, phosphate buffer (1 ml, pH 7,4) was used to wet the gel; the dialysis membrane was only immersed in the phosphate buffer that served as a receiving enclosure. At 37 • C, the receiving section (100 rpm, Remi, India) was removed magnetically. Samples of (1 ml) were removed periodically from the reception area. A spectrophotometer of 248nm (Shi-madzu1800) was used to calculate the amount of brimonidine maleate released by the nano lipid in situ gel. Following through sample withdrawal, the reception bay was illed with a quantity equal to the phosphate buffer. (Shashank et al., 2015;Nayak and Srinivasa, 2017a)

Accelerated stability studies
For a short-term, accelerated stability test at 4ºC±2ºC and 27ºC±2ºC engineered nano lipid dispersion that had higher trapping ef iciency was put in vials and screened with aluminium foil as amended by the international harmonization guidelines conference. Every 90 days product content samples were analyzed. (Nayak and Srinivasa, 2017b)

Study of interaction of the drug with excipients used in the formulation
The FTIR spectrum studies of brimonidine maleate pure drug and drugs loaded nano lipid gel were analyzed. The primary functional group's peaks of brimonidine maleate present in loaded nano lipid gel were intact and were present (Table 3 & Figures 1  and 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.

Percentage drug entrapment ef iciency of Nanolipid in situ formulations
The nature of lipids played a signi icant role in drug entrapment ef iciency. The entrapment ef iciency of the system was calculated as a ratio of the amount of drug entrapped by the system to the amount of drug taken, expressed in percentage. The entrapment ef iciency was within the range of 67.20% to 97.3% for Brimonidine maleate loaded insitu gel formulations. F1 entrapment ef iciency was found to be 97.3% and shown maximum when compared with other formulations (Table 4 & Figure 3).

Percentage drug content of Nanolipid in situ formulations
Brimonidine maleate loaded insitu gel formulations were analyzed for drug content spectrophotometrically at 248nm. Brimonidine maleate loaded insitu gel formulations exhibited relatively uniform drug content. The drug content was between 65.69% and 96.36% for all formulations, as shown in [ Table 5].
The F6 formulation showed maximum drug content of 96.36%.

Visual appearance and pH of Nanolipid insitu formulations
For the nature of some particular matter, visual appearance and clarity were observed. In the corneal membrane, an acidic or alkaline solution induces irritation. The pH of nanoparticle insitu gel was detected by using digital pH meter. Nanolipid in-situ gel pH range lies between 4.9-7.1 pH (Table 6). Nanolipid in-situ gel shows maximum pH 7.1 for F6 Brimonidine maleate loaded insitu gel formulations the pH of the reported formulations was non-irritable to the eye. This re lects the gel is not harmful to the eye surface.

The gelling capacity of Nanolipid in situ formulations
The gelling capacity was determined by freshly prepared simulated tear luid (STF  Figure 4).

Stability studies of Nanolipid in situ formulations
Stability studies of optimized BF6 Brimonidine maleate insitu Gel were conducted for three months at 4±2 • C and 27±2 • C. For precipitation, the formulations were visually tested. The drug content was measured for three months every 30 days. The physical appearance of the solution was found to be unchanged. The drug quality of these formulations was analyzed, and there were small variations between them at different temperatures, as shown in (Table 9). During the whole study, nano lipid in situ formulations maintained good stability.

CONCLUSIONS
We had formulated different formulation of nanoparticle drug delivery system they are nano lipid incorporate in suit gels with drug Brimonidine Maleate for ocular therapy. The formulated nanoparticles are characterized and evaluated. In nano lipid in suit gel formulations of Brimonidine Maleate can able to overcome precorneal and nasolacrimal drainage disadvantages. The formulations showed excellent drug loading capacity. The formulation was stable, nonirritant and release drugs in sustain manner form the gel formulation. It was inally concluded from the above work that formulation F1 has a maximum entrapment ef iciency of 97.30% and drug content of about 96.36% for formulation F6. F1 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 99% for 10 hrs.