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\def\RunningAuthor{Mohd Azharuddin et al.}
\firstPage{6837}
\articleType{Original Article}
\receivedDate{04 Aug 2020}
\acceptedDate{09 Sep 2020}
\revisedDate{06 Sep 2020}
\journalVolume{2020, 11}
\journalIssue{4}
\journalDoi{ijrps.v11i4.3654}
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\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{\textbf{Design development and evaluation of novel ophthalmic nano lipid \textit{in situ} gel-forming solution using timolol hydrochloride}}
\author{Mohd~Azharuddin\textsuperscript{*}\textsuperscript{1},  
            Theivendren~Panner~Selvam\textsuperscript{2},  
            Maya~Sharma\textsuperscript{3}, 
            Jayesh~Dwivedi\textsuperscript{4}~\\[5pt]{\textsuperscript{1}Pacific Academy of Higher Education and Research University, Udaipur, Rajasthan, India}~\\{\textsuperscript{2}Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy, Elayampalayam, Namakkal, Tamil Nadu, India}~\\{\textsuperscript{3}Department of Pharmaceutical Chemistry, Pacific College of Pharmacy, PAHER University, Udaipur, Rajasthan, India}~\\{\textsuperscript{4}Pacific College of Pharmacy, PAHER University, Udaipur, Rajasthan, India}}

\begin{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 film 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 efficiency 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 finally concluded from the present work that formulations prepared were more suitable and had good patient compliance compared to the eye drops.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Timolol hydrochloride,\newline in situ gel,\newline entrapment efficiency,\newline in-vitro release,\newline gelation study,\newline ocular irritation
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Mohd~Azharuddin~\\ Phone:\ +919886447725~\\  Email:\ azhartvmcop@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{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 significant 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 \unskip~\citep{883287:20304854}.

Various stimuli can form gels theses includes 


  
  \begin{enumerate}
  \item \relax Physical stimuli: Change in temperature, electric fields, light, pressure, sound, and magnetic fields.
  \item \relax Chemical stimuli: Change in pH and ion activation from biological fluids.
  \item \relax Biological or biochemical stimuli: Change in glucose level.
  \end{enumerate}
  From the above various stimuli only pH, ion activated, and temperature stimuli can be used for designing of ophthalmic drug delivery system. 

Types of \textit{in situ} ophthalmic gel are 


  
  \begin{enumerate}
  \item \relax \textit{In situ} gelling based on change in pH
  \item \relax \textit{In situ} gelling based on Temperature Modulation
  \item \relax Ion-activated in situ Gellation \unskip~\citep{883287:20304859}.
  \end{enumerate}
  In the present research work, timolol hydrochloride nano lipid \textit{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 fluid and adjust pH) were added to the gel batches in sufficient quantity.
    
\section{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.

\textbf{Study of interaction of the drug with excipients used in the formulation\ensuremath{^{}}}

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 \textit{in situ} gel \unskip~\citep{883287:20304864}.

\textbf{Manufacture of nano lipids \textit{in situ} gel }

\textbf{Formulation of nano lipids} \textit{\space }

Film hydration technique was used for the preparation of nano lipids. In round bottom flask mixture of vesicles like lecithin and cholesterol were dissolved in a volatile organic solvent (methanol). The rotary evaporator was rotated at 60\text{\textordmasculine}C for 45 min. The organic solvent is removed with gentle agitation, leaving a thin film 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 flask at room temperature and followed by sonication for 30 min. Nanolipid solution was cooled by kept in 4-8\text{\textordmasculine}C in the freezer.

\textbf{Formulation of nano lipid in-situ gel\ensuremath{^{}}}

Nanolipid \textit{in situ} gels were prepared purely based on drug entrapment efficiency and morphology. The batch which gave maximum entrapment and good surface morphology were selected for preparation of \textit{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 fluid were added to the gel batches in sufficient quantity to adjust the pH (Table~\ref{tw-5acfdecc7cbe}) \unskip~\citep{883287:20304866}.

\textbf{Drug entrapment efficiency}

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 fluid and was analyzed by UV spectrophotometric method. 

Drug entrapment efficiency was calculated by using the equation. The amount of timolol hydrochloride encapsulated in the nano lipid was determined by using EE = (Total drug concentration {\textendash} free drug concentration) / total drug concentration \ensuremath{\times} 100 where, EE is entrapment efficiency (\%) \unskip~\citep{883287:20304858}.

\textbf{Estimation of drug content}

50 mg of equivalent nano lipid suspension was taken into a standard volumetric flask. 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 fluid 7.2. The absorbance was measured at 295 nm for timolol hydrochloride, and drug content was calculated with the help of calibration curve \unskip~\citep{883287:20304862,883287:20304856}.

\textbf{Visual appearance and pH\ensuremath{^{}}}

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 \unskip~\citep{883287:20304853}.

\textit{\textbf{In vitro}} \textbf{ gellation study}\ensuremath{^{\textbf{\space }}}

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 fluid (STF) pH 7.2 and equilibrated at 37\text{\textordmasculine}C. Than gel formation was observed and time required for gelation and dissolution of the gel formed was noted \unskip~\citep{883287:20304855}.

\textbf{Viscosity study\ensuremath{^{}}}

Brookfield viscometer was used to determine the viscosity of the formulations by using CPE - 42 spindle at 10, 20, 50 \&100 revolutions per min \unskip~\citep{883287:20304861,883287:20304860}.

\textit{\textbf{In vitro}} \textbf{ drug } \textbf{release of nano lipid \textit{in situ} gel}\ensuremath{^{\textbf{\space }}}

\textit{In vitro }release studies of timolol hydrochloride nano lipid, the \textit{in-situ} gel was performed at 37\text{\textordmasculine}C using stimulated tear fluid (pH 7.2) as the release medium. 

Nanolipid \textit{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 fluid (1 ml, pH 7.2) was added in the reservoir compartment. 

The receiving compartment was stirred magnetically (100 rpm) at 37\text{\textordmasculine}C. At regular interval, 1 ml of samples were withdrawn from the receiving compartment, and the amount of timolol hydrochloride released from \textit{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 \unskip~\citep{883287:20304857}.

\textbf{Ocular irritation studies\ensuremath{^{}}}

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 (27\text{\textordmasculine}C){\textemdash}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 \unskip~\citep{883287:20304863}.

\textbf{Accelerated stability studies\ensuremath{^{}}}

The optimized nano lipid dispersion, which had higher entrapment efficiency subjected to short term accelerated stability study at 4\text{\textordmasculine}C \ensuremath{\pm} 2\text{\textordmasculine}C and 27\text{\textordmasculine}C \ensuremath{\pm} 2\text{\textordmasculine}C as per modified ICH guidelines. Samples were analyzed for drug content \unskip~\citep{883287:20304865}.
    
\section{Results and Discussion}
\textbf{Study of interaction of the drug with excipients used in the formulation}

The FTIR spectrum studies of Timolol hydrochloride drug and \textit{in-situ} gel were analyzed. Primary functional groups peaks \{3315.52 (NH\ensuremath{_{str}}), 3049.90 (Ar-CH\ensuremath{_{str}}), 1615.26 (C=N) and 808.42 (Br)\} of timolol hydrochloride were present in loaded \textit{in-situ} gel prepared (Table~\ref{tw-6ce978c2b0e8}, Table~\ref{tw-ad68509664d2}, Figure~\ref{f-39e77fe1d027} and Figure~\ref{f-3bc4e09dafaa}). 

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.\textbf{\space }


\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Formulation of Timolol hydrochloride loaded nanolipid \textit{in-situ} gels}} }
\label{tw-5acfdecc7cbe}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Ingredients & \cAlignHack TF1 & \cAlignHack TF2 & \cAlignHack TF3 & \cAlignHack TF4 & \cAlignHack TF5 & \cAlignHack TF6\\
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Timolol hydrochloride  \% w/v &
  \cAlignHack 0.05 &
  \cAlignHack 0.05 &
  \cAlignHack 0.05 &
  \cAlignHack 0.05 &
  \cAlignHack 0.05 &
  \cAlignHack 0.05\\
Lecithin \% w/v &
  \cAlignHack 0.05 &
  \cAlignHack 0.05 &
  \cAlignHack 0.1 &
  \cAlignHack 0.05 &
  \cAlignHack 0.15 &
  \cAlignHack 0.2\\
Cholesterol \% v/v &
  \cAlignHack 0.05 &
  \cAlignHack 0.1 &
  \cAlignHack 0.05 &
  \cAlignHack 0.15 &
  \cAlignHack 0.1 &
  \cAlignHack 0.05\\
Methanol \% v/v &
  \cAlignHack 7.5 &
  \cAlignHack 7.5 &
  \cAlignHack 7.5 &
  \cAlignHack 7.5 &
  \cAlignHack 7.5 &
  \cAlignHack 7.5\\
Water \% v/v &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 10 &
  \cAlignHack 10\\
HPMC K 15M \% w/v &
  \cAlignHack 0.2 &
  \cAlignHack 0.2 &
  \cAlignHack 0.4 &
  \cAlignHack 0.4 &
  \cAlignHack 0.3 &
  \cAlignHack 0.2\\
Carbopol 940 \% w/v &
  \cAlignHack 0.2 &
  \cAlignHack 0.4 &
  \cAlignHack 0.2 &
  \cAlignHack 0.4 &
  \cAlignHack 0.2 &
  \cAlignHack 0.3\\
EDTA \% w/v &
  \cAlignHack 0.1 &
  \cAlignHack 0.1 &
  \cAlignHack 0.1 &
  \cAlignHack 0.1 &
  \cAlignHack 0.1 &
  \cAlignHack 0.1\\
Benzolkonium Chloride \% v/v &
  \cAlignHack 0.01 &
  \cAlignHack 0.01 &
  \cAlignHack 0.01 &
  \cAlignHack 0.01 &
  \cAlignHack 0.01 &
  \cAlignHack 0.01\\
Sodium chloride \% w/v &
  \cAlignHack 0.9 &
  \cAlignHack 0.9 &
  \cAlignHack 0.9 &
  \cAlignHack 0.9 &
  \cAlignHack 0.9 &
  \cAlignHack 0.9\\
Stimulated Tear Fluid \% v/v &
  \cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100 &
  \cAlignHack 100\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{FTIR spectra data of Timolol hydrochloride }} }
\label{tw-6ce978c2b0e8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5207\linewidth-2\tabcolsep}p{\dimexpr.4793\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack IR (KBr)  & \cAlignHack Peaks cm-1 \\
\tblmidrule 
\cAlignHack NHstr  &
  \cAlignHack 3315.52 \\
\cAlignHack Ar-CHstr &
  \cAlignHack 3049.90\\
\cAlignHack C=N  &
  \cAlignHack 1615.26\\
\cAlignHack Br &
  \cAlignHack 808.42\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{FTIR spectra data of formulated Timolol hydrochloride \textit{in-situ } gel}} }
\label{tw-ad68509664d2}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5021\linewidth-2\tabcolsep}p{\dimexpr.4979\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack IR (KBr)  & \cAlignHack Peaks cm-1 \\
\tblmidrule 
\cAlignHack NHstr  &
  \cAlignHack 3366.59 \\
\cAlignHack Ar-CHstr &
  \cAlignHack 3043.61\\
\cAlignHack OHstr  &
  \cAlignHack 2982.78\\
\cAlignHack C=N  &
  \cAlignHack 1621.23 \\
\cAlignHack Br &
  \cAlignHack 834.18\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Entrapment Efficiency of Timolol hydrochloride loaded nano lipid \textit{in-situ} gels}} }
\label{tw-d935c4505ccf}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5332\linewidth-2\tabcolsep}p{\dimexpr.4668\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Formulation  & \cAlignHack Entrapment Efficiency \% \\
\tblmidrule 
\cAlignHack TF1  &
  \cAlignHack 95.27 \ensuremath{\pm} 1.707 \\
\cAlignHack TF2  &
  \cAlignHack 97.30 \ensuremath{\pm} 1.326 \\
\cAlignHack TF3  &
  \cAlignHack 73.40 \ensuremath{\pm} 0.604 \\
\cAlignHack TF4  &
  \cAlignHack 79.96 \ensuremath{\pm} 0.356 \\
\cAlignHack TF5  &
  \cAlignHack 66.28 \ensuremath{\pm} 1.896 \\
\cAlignHack TF6  &
  \cAlignHack 93.43 \ensuremath{\pm} 1.506 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{\space }


\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Drug Content Estimation of Timolol hydrochloride loaded nano lipid \textit{in-situ } gels}} }
\label{tw-7b5d811b3a74}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1661\linewidth-2\tabcolsep}p{\dimexpr.4939\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No & \cAlignHack Formulations  & \cAlignHack Drug content\% \\
\tblmidrule 
\cAlignHack 1  &
  \cAlignHack TF1  &
  \cAlignHack 81.24\ensuremath{\pm}0.724 \\
\cAlignHack 2  &
  \cAlignHack TF2  &
  \cAlignHack 97.67\ensuremath{\pm}0.637 \\
\cAlignHack 3  &
  \cAlignHack TF3  &
  \cAlignHack 66.69\ensuremath{\pm}1.367 \\
\cAlignHack 4  &
  \cAlignHack TF4  &
  \cAlignHack 88.09\ensuremath{\pm}0.545 \\
\cAlignHack 5  &
  \cAlignHack TF5  &
  \cAlignHack 76.65\ensuremath{\pm}0.756 \\
\cAlignHack 6  &
  \cAlignHack TF6  &
  \cAlignHack 88.43\ensuremath{\pm}1.009 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Visual Appearance and pH of Timolol hydrochloride nano lipid \textit{in-situ} gel formulations}} }
\label{tw-8d6116b06560}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.13590000000000002\linewidth-2\tabcolsep}p{\dimexpr.3641\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No & \cAlignHack Formulations & \cAlignHack Visual appearance & \cAlignHack pH\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack TF1 &
  \cAlignHack Clear &
  \cAlignHack 4.6 \ensuremath{\pm} 0.123\\
\cAlignHack 2 &
  \cAlignHack TF2 &
  \cAlignHack Clear &
  \cAlignHack 4.2 \ensuremath{\pm} 0.354\\
\cAlignHack 3 &
  \cAlignHack TF3 &
  \cAlignHack Cloudy &
  \cAlignHack 4.6 \ensuremath{\pm} 0.345\\
\cAlignHack 4 &
  \cAlignHack TF4 &
  \cAlignHack Clear &
  \cAlignHack 4.1 \ensuremath{\pm} 0.032\\
\cAlignHack 5 &
  \cAlignHack TF5 &
  \cAlignHack Cloudy &
  \cAlignHack 4.8 \ensuremath{\pm} 0.867\\
\cAlignHack 6 &
  \cAlignHack TF6 &
  \cAlignHack Clear &
  \cAlignHack 4.3 \ensuremath{\pm} 0.323\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Gelling Capacity of Timolol hydrochloride nano lipid \textit{in-situ} gel formulations}} }
\label{tw-221921f65ace}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1868\linewidth-2\tabcolsep}p{\dimexpr.41510000000000005\linewidth-2\tabcolsep}p{\dimexpr.3981\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No & \cAlignHack Formulations  & \cAlignHack Gellation capacity \\
\tblmidrule 
\cAlignHack 1  &
  \cAlignHack TF1 &
  \cAlignHack +++ \\
\cAlignHack 2  &
  \cAlignHack TF2 &
  \cAlignHack ++++ \\
\cAlignHack 3  &
  \cAlignHack TF3 &
  \cAlignHack +++ \\
\cAlignHack 4  &
  \cAlignHack TF4 &
  \cAlignHack ++++ \\
\cAlignHack 5  &
  \cAlignHack TF5 &
  \cAlignHack +++ \\
\cAlignHack 6  &
  \cAlignHack TF6 &
  \cAlignHack ++++ \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Viscosity Study of Timolol hydrochloride nano lipid \textit{in-situ} gel formulations}} }
\label{tw-5ce8deecc9b3}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1958\linewidth-2\tabcolsep}p{\dimexpr.1257\linewidth-2\tabcolsep}p{\dimexpr.1412\linewidth-2\tabcolsep}p{\dimexpr.1353\linewidth-2\tabcolsep}p{\dimexpr.12690000000000001\linewidth-2\tabcolsep}p{\dimexpr.14239999999999998\linewidth-2\tabcolsep}p{\dimexpr.1327\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Angular velocity  & \multicolumn{6}{p{\dimexpr(.8041999999999999\linewidth-2\tabcolsep)}}{\cAlignHack Viscosity(CPS)}\\
\rowcolor{kwdboxcolor}\cAlignHack (rpm)  & \cAlignHack TF1 & \cAlignHack TF2 & \cAlignHack TF3  & \cAlignHack TF4  & \cAlignHack TF5  & \cAlignHack TF6 \\
\tblmidrule 
\cAlignHack 10  &
  \cAlignHack 527\ensuremath{\pm}1.456  &
  \cAlignHack 905\ensuremath{\pm}1.845  &
  \cAlignHack 713\ensuremath{\pm}1.234  &
  \cAlignHack 768\ensuremath{\pm}0.389  &
  \cAlignHack 702\ensuremath{\pm}0.456  &
  \cAlignHack 645\ensuremath{\pm}1.346 \\
\cAlignHack 20  &
  \cAlignHack 485\ensuremath{\pm}1.678  &
  \cAlignHack 807\ensuremath{\pm}0.456  &
  \cAlignHack 612\ensuremath{\pm}0.345  &
  \cAlignHack 678\ensuremath{\pm}1.450  &
  \cAlignHack 605\ensuremath{\pm}0.950  &
  \cAlignHack 421\ensuremath{\pm}1.945 \\
\cAlignHack 50  &
  \cAlignHack 323\ensuremath{\pm}1.543  &
  \cAlignHack 708\ensuremath{\pm}1.567  &
  \cAlignHack 305\ensuremath{\pm}0.456  &
  \cAlignHack 467\ensuremath{\pm}0.345  &
  \cAlignHack 382\ensuremath{\pm}1.423  &
  \cAlignHack 389\ensuremath{\pm}1.586 \\
\cAlignHack 100  &
  \cAlignHack 256\ensuremath{\pm}1.789  &
  \cAlignHack 596\ensuremath{\pm}0.213  &
  \cAlignHack 189\ensuremath{\pm}0.478  &
  \cAlignHack 285\ensuremath{\pm}0.386  &
  \cAlignHack 199\ensuremath{\pm}0.765  &
  \cAlignHack 256\ensuremath{\pm}0.945 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Cumulative percentage drug release profile of }\textbf{Timolol hydrochloride} \textbf{ nano lipid \textit{in situ} gel formulations}} }
\label{tw-857d6629067a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.12459999999999999\linewidth-2\tabcolsep}p{\dimexpr.1412\linewidth-2\tabcolsep}p{\dimexpr.14109999999999998\linewidth-2\tabcolsep}p{\dimexpr.13879999999999995\linewidth-2\tabcolsep}p{\dimexpr.14600000000000001\linewidth-2\tabcolsep}p{\dimexpr.14830000000000002\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Time(hrs)  & \cAlignHack TF1 (\%)  & \cAlignHack TF2 (\%)  & \cAlignHack TF3 (\%)  & \cAlignHack TF4 (\%)  & \cAlignHack TF5 (\%)  & \cAlignHack TF 6 (\%) \\
\tblmidrule 
\cAlignHack 0.5  &
  \cAlignHack 7.42\ensuremath{\pm}0.041 &
  \cAlignHack 6.25\ensuremath{\pm}0.458 &
  \cAlignHack 7.98\ensuremath{\pm}0.895 &
  \cAlignHack 18.07\ensuremath{\pm}0.256 &
  \cAlignHack 16.05\ensuremath{\pm}0.546  &
  \cAlignHack 13.07\ensuremath{\pm}0.405 \\
\cAlignHack 1  &
  \cAlignHack 37.04\ensuremath{\pm}0.506 &
  \cAlignHack 18.42\ensuremath{\pm}0.356 &
  \cAlignHack 22.13\ensuremath{\pm}1.207 &
  \cAlignHack 41.01\ensuremath{\pm}0.645 &
  \cAlignHack 38.45\ensuremath{\pm}0.305  &
  \cAlignHack 30.04\ensuremath{\pm}0.549 \\
\cAlignHack 2  &
  \cAlignHack 51.16\ensuremath{\pm}3.061 &
  \cAlignHack 41.32\ensuremath{\pm}3.457 &
  \cAlignHack 39.30\ensuremath{\pm}1.264 &
  \cAlignHack 53.0\ensuremath{\pm}0.856 &
  \cAlignHack 43.07\ensuremath{\pm}1.805  &
  \cAlignHack 35.8 \ensuremath{\pm}1.234 \\
\cAlignHack 4  &
  \cAlignHack 65.18\ensuremath{\pm}1.987 &
  \cAlignHack 51.67\ensuremath{\pm}0.87 &
  \cAlignHack 54.62\ensuremath{\pm}1.089 &
  \cAlignHack 67.46\ensuremath{\pm}0.309 &
  \cAlignHack 57.98\ensuremath{\pm}0.895  &
  \cAlignHack 52.05\ensuremath{\pm}1.867 \\
\cAlignHack 6  &
  \cAlignHack 82.30\ensuremath{\pm}1.640 &
  \cAlignHack 69.57\ensuremath{\pm}1.23 &
  \cAlignHack 68.45\ensuremath{\pm}0.956 &
  \cAlignHack 85.59\ensuremath{\pm}1.006 &
  \cAlignHack 80.03\ensuremath{\pm}0.856  &
  \cAlignHack 70.90\ensuremath{\pm}1.867 \\
\cAlignHack 10  &
  \cAlignHack 98.50\ensuremath{\pm}1.45 &
  \cAlignHack 84.29\ensuremath{\pm}1.760 &
  \cAlignHack 86.98\ensuremath{\pm}1.079 &
  \cAlignHack 85.82\ensuremath{\pm}1.176 &
  \cAlignHack 84.05\ensuremath{\pm}1.265  &
  \cAlignHack 80.03\ensuremath{\pm}0.466 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Grading of Ocular Lesions}} }
\label{tw-7da542f9fe6e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.13919999999999998\linewidth-2\tabcolsep}p{\dimexpr.7116\linewidth-2\tabcolsep}p{\dimexpr.14920000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Cornea}\\
\tblmidrule 
1 &
  No ulceration or opacity &
  0\\
2 &
  Slight dulling of opacity &
  1\\
3 &
  Easily discernible translucent area &
  2\\
4 &
  Nacrous area: no details of iris visible &
  3\\
5 &
  Opaque cornea; iris not discernible through the opacity  &
  4\\
\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Iris}\\
1 &
  Normal &
  0\\
2 &
  Congestion, swelling , iris reactive to light (a sluggish reaction) &
  1\\
3 &
  Hemorrhage or no reaction to light  &
  2\\
\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Conjuctivea}\\
1 &
  Normal &
  0\\
2 &
  Some blood vessels hyperaemic (injected) &
  1\\
3 &
  Individual vessels not easily discernible  &
  2\\
4 &
  Diffuse beefy red  &
  3\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Ocular Irritation Study} }
\label{tw-326fd51f5aac}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.15990000000000002\linewidth-2\tabcolsep}p{\dimexpr.5001\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S.No & \cAlignHack Parts of Eye & \cAlignHack TF4\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack Cornea  &
  \cAlignHack  normal \\
\cAlignHack 2 &
  \cAlignHack Iris  &
  \cAlignHack  normal \\
\cAlignHack 3 &
  \cAlignHack Conjunctivae  &
  \cAlignHack  normal \\
\cAlignHack 4 &
  \cAlignHack Total  &
  \cAlignHack  normal \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Stability Data of OptimizedFormulation} }
\label{tw-047938fcdf07}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1129\linewidth-2\tabcolsep}p{\dimexpr.2071\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{6}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Drug content}\\
\rowcolor{kwdboxcolor} & \cAlignHack Storage Conditions & \cAlignHack Initial & \cAlignHack 1 month & \cAlignHack 2 months & \cAlignHack 3 months\\
\tblmidrule 
TF2 &
  \cAlignHack 4\ensuremath{^\circ}C\ensuremath{\pm}2\ensuremath{^\circ}C &
  \cAlignHack 97.23\ensuremath{\pm}1.517 &
  \cAlignHack 96.04\ensuremath{\pm}1.567 &
  \cAlignHack 95.37\ensuremath{\pm}1.231 &
  \cAlignHack 94.34\ensuremath{\pm}1.491\\
 &
  \cAlignHack 27\ensuremath{^\circ}C\ensuremath{\pm}2\ensuremath{^\circ}C  &
  \cAlignHack 97.23\ensuremath{\pm}0.438 &
  \cAlignHack 95.87\ensuremath{\pm}1.349 &
  \cAlignHack 95.07\ensuremath{\pm}1.519 &
  \cAlignHack 94.06\ensuremath{\pm}1.076\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/1c1da598-4b84-4f0a-8b19-9fd4a92b050c-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/1c1da598-4b84-4f0a-8b19-9fd4a92b050c-upicture1.png}{\includegraphics{images/1c1da598-4b84-4f0a-8b19-9fd4a92b050c-upicture1.png}}{}
\makeatother 
\caption{\boldmath {FTIR spectra of Timolol hydrochloride}}
\label{f-39e77fe1d027}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/291d55f9-e8bc-4da0-8b60-b1de4f4d5a7d-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/291d55f9-e8bc-4da0-8b60-b1de4f4d5a7d-upicture2.png}{\includegraphics{images/291d55f9-e8bc-4da0-8b60-b1de4f4d5a7d-upicture2.png}}{}
\makeatother 
\caption{\boldmath {FTIR spectra of formulated Timolol hydrochloride \textit{in-situ} gel}}
\label{f-3bc4e09dafaa}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/ea811b71-dbc3-4022-befc-ded7bfa235ca-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/ea811b71-dbc3-4022-befc-ded7bfa235ca-upicture3.png}{\includegraphics{images/ea811b71-dbc3-4022-befc-ded7bfa235ca-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Comparative entrapment efficiencies of Timolol hydrochloride loaded nanolipid \textit{in-situ }gels}}
\label{f-a635dd78804c}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/bd85c6aa-6712-4691-be6b-dd093778253d-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/bd85c6aa-6712-4691-be6b-dd093778253d-upicture4.png}{\includegraphics{images/bd85c6aa-6712-4691-be6b-dd093778253d-upicture4.png}}{}
\makeatother 
\caption{\boldmath {\textit{In-vitro} drug releases of Timolol hydrocholride nanolipid \textit{in-situ} gels formulations}}
\label{f-12221e89fe3c}
\end{figure}
\egroup
\textbf{Drug entrapment efficiency}

The entrapment efficiency was calculated, and it is expressed in term of percentage. Entrapment efficiency was within the range of 73.40\% to 97.30\%  for timolol hydrochloride loaded \textit{in-situ} gel formulations. 

TF2 formulation showed maximum entrapment efficiency of 97.30\% when compared with other formulations (Table~\ref{tw-d935c4505ccf} and Figure~\ref{f-a635dd78804c}). 

\textbf{Estimation of drug content}

Timolol hydrochloride loaded \textit{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~\ref{tw-7b5d811b3a74}). The TF2 formulation showed maximum drug content of about 97.67\%.

\textbf{Visual appearance and pH}

The presence of any particular matter was observed visually. Timolol hydrochloride nano lipid \textit{in situ} gel pH range lies between 4.1- 4.8 pH (Table~\ref{tw-8d6116b06560}). Nanolipid \textit{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.

\textbf{In-vitro} \textbf{ gellation study}

Freshly prepared simulated tear fluid 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\textbf{\space }as shown in Table~\ref{tw-221921f65ace}.

\textbf{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 highest drug release. Intermediate viscosity of formulations has shown maximum retardation of drug release due to the dense nature of the polymers. Carbopol 940 and HPMC K-15M as polymer system have contributed majorly towards building viscosity of the formulation. Prepared nano lipid \textit{in-situ} gel formulations viscosity was in the range between 256 - 905 cps, as shown in Table~\ref{tw-5ce8deecc9b3}.

\textit{\textbf{In-vitro}}  \textbf{release of nano lipid \textit{in situ} gel}

The timolol hydrochloride nano lipid \textit{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 \textit{in vitro} release profiles, TF2 was selected as optimized formulations (Table~\ref{tw-857d6629067a} and Figure~\ref{f-12221e89fe3c}). 

\textbf{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~\ref{tw-7da542f9fe6e} and Table~\ref{tw-326fd51f5aac}.

\textbf{Accelerated stability studies}

The stability studies of optimized formulation TF4 timolol hydrochloride nano lipid \textit{in situ} gel formulations was determined at 4\ensuremath{^\circ}C \ensuremath{\pm} 2\ensuremath{^\circ}C and 27\ensuremath{^\circ}C \ensuremath{\pm} 2\ensuremath{^\circ}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~\ref{tw-047938fcdf07}. 
    
\section{Conclusion}
It was concluded from the above work that formulation TF2 has a maximum entrapment efficiency 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.
\section*{Acknowledgement}The author is thankful to the management and principal of TVM college of Pharmacy, Bellary, Karnataka for providing the necessary facilities to carry out the research work. 

\textbf{Funding support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.
    

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