Determination of Mucoadhesive behaviour of Timolol maleate liquid crystalline cubogel by different Techniques


Ankit Acharya*, Prakash Goudanavar, Vinay C H

Department of Pharmaceutics, Sri Adichunchanagiri College of Pharmacy,

B.G. Nagara-571448, Karnataka, India

*Corresponding Author E-mail:



Glaucoma is an ocular disease characterized by the increasing of the intraocular pressure (IOP), optic nerve head changing and decreasing of retinal sensitivity that ultimately lead to the loss of vision. Glaucoma is the second most common cause of blindness in the world. The present study involved formulation and evaluation of mucoadhesive Timolol maleate loaded, liquid crystalline cubogel for the treatment of glaucoma. Firstly cubosome was prepared by top down technique using 10:1 ratio of lipid (glycerol monooleate) and surfactant (poloxamer 407). Cubosome was then converted into cubogel by dispersing it into in-situ gel formulation. Cubogel formulations were then evaluated for pH, drug content, viscosity and mucoadhesive properties. Mucoadhesive strength was determined by ultracentrifugation and turbidity method. Photomicroscope showed bilayer structure cubosome. Cubosomal dispersion was milky white in appearance and having particle size of 181 nm. The drug content was found to be 97.28±0.048% and 98.95±0.041% respectively, for CG1 and CG2 formulation. Prepared cubogel formulation showed significantly higher mucoadhesive behaviour compared to cubosomal dispersion. Finally, it can be concluded that cubogel could increase the residence time of drug in the eye by its mucoadhesive property and reduce number of application of drugs. Hence, it can be beneficial in case of glaucoma as required long term treatment.


KEYWORDS: Glaucoma, Timolol maleate, cubogel, mucoadhesive property.




Commonly used conventional eye drops show low ocular bioavailability because of constant lachrymal secretion and rapid nasolachrymal drainage. Normal drainage of an instilled dose commences immediately on instillation and is essentially completed within 5 min1. Typically, ophthalmic bioavailabilities less than 5% are achieved due to the short precorneal residence times of ophthalmic solutions.



Consequently, there is a need for frequent instillation of concentrated solutions to achieve the desired therapeutic effect. To overcome these problems various ophthalmic formulations, such as viscous solutions, ointments, gels, nanoparticles or polymeric inserts have been investigated2. The corneal contact time has been increased to varying degrees by these vehicles. But, they have not been unanimously accepted, because of blurred vision (e.g. ointments) or lack of patient compliance (e.g. inserts). As a result, good ocular bioavailability following topical delivery of a drug to the eye remains a challenge and yet to be resolved3.


In order to improve ocular bioavailability many research on drug reservoir system have been made. These reservoir systems are carrier of drug molecules and the controller of the drug release pattern. Cubosomes will be used as a drug reservoir system as they are successfully used for ocular drug delivery system. Cubosomes are cubic shape liquid crystals dispersion4.


They have unique nanostructure of a highly twisted, uninterrupted lipid bilayer and two congruent non-transecting water channels. Furthermore, the microstructure of cubosomes is similar to that of biological membranes. This enables the lipid carriers to fuse with the lipid bilayers of the corneal epithelial cells. Therefore, cubosomes are outstanding candidates for the ocular drug delivery system according to their physicochemical features5.


However, cubosome is a low viscosity liquid form product and its particle is susceptible to aggregation. Due to low viscosity it does not have sufficient contact time of the drug in the eye, so the method needs to be improved. One strategy used to decrease the precorneal drainage rate of cubosomes is dispersing the cubosomes into an ion-sensitive (gellan gum), temperature dependent (chitosan) and pH dependent (carbopol) polymer. Decrease in precorneal drainage of installed dosage form is due to mucoadhesive property of such polymer. The advantages of mucoadhesive drug delivery systems include the prolongation of residence time of the dosage form at the site of absorption leading to enhanced absorption and hence the therapeutic efficacy of the drug6.


In this study Timolol maleate (TM) was selected as model drug. Timolol maleate is a non-selective beta-blocker, used in the treatment of glaucoma. Glaucoma is the second most common cause of blindness in the world. It affects more than 70 million people worldwide7. Timolol was selected because of more rapid onset, better tolerance, and fewer side effects than other drugs in the same class. Timolol maleate lowers intra ocular pressure (IOP) by reducing the production of the aqueous humor through blocking the sympathetic nerve endings in the ciliary epithelium. The conventional eye drops of Timolol maleate is available at low-cost, convenient to use, and it significantly lower IOP8. However, due to the unique anatomical structure of the eye, the corneal epithelial barrier function limits drug absorption from the lacrimal fluid into the anterior chamber after eye drop administration. Low bioavailability (<5%) of TM eye drops can lead to ineffective therapy. Topically administrated TM eye drops have poor corneal permeability, and most of the eye drops drain through the nasolacrimal duct. In order to minimize such problems mucoadhesive cubogel formulation of TM was formulated and evaluated for its mucoadhesive property9.



Pure drug Timolol maleate, poloxomer 407 and glycerol monooleate (GMO) were purchase from Yarrow Chem products, Mumbai, India.  Carbopol 940p, gellan gum and chitosan were procured from Himedia, Mumbai. All other reagents used were analytical grade. 


Preparation of Timolol maleate cubosome:

Timolol maleate-loaded cubosome was prepared by top down techniques. The composition of cubosome is showed in table 1.  Glycerol monooleate (6ml) and poloxamer 407 (0.6 gm) were accurately weigh and melted at 60oC in a hot water bath. Then glycerol was added to melted poloxamer and GMO dispersion. Drug was dissolved in 3 ml of Millipore water and gradually added to above dispersion and vortex mixed for 2 minute. After equilibration for 10 hours at room temperature, the cubic phase gel was formed. Remaining quantity of water was added to cubic gel and gel was fragmented for 10 minutes by intermittent probe sonication. Afterward, the resulting milky coarse dispersion of cubosome was formed10.


Preparation of Timolol maleate based cubogel:

The composition of cubosome loaded Timolol maleate in-situ gel, i.e. cubogel is depicted in Table 1. In-situ gel formulation was prepared by cold method using gellan gum and chitosan as polymers and carbopol p934 as co-polymer. Cubosome dispersion was sprinkled into previously prepared in-situ gel and stirred using magnetic stirrer. Methylparaben (0.1%) and NaCl (0.45%) were used as preservatives and tonicity adjusting agents, respectively11.


Table 1: Composition of Timolol maleate cubogel

Ingredients (%)




Timolol maleate








Poloxamer 407




Gellan gum








Carbopol p934




Methyl paraben








Distilled water




A6 is cubosome; CG1 and CG2 are cubogel formulation


Characterization of cubosome:

Visual inspection:

Prepared cubosome dispersion was visually inspected for appearance and phase separation12.


Determination of Particle size and zeta potential:

Particle size, polydispersity index, and zeta potential were determined by using Zetasizer Nano ZS (Malvern Instruments, UK). The instrument was equipped with dynamic light scattering particle size analyzer at a wavelength of 635 nm and a fixed scattering angle of 90°. The values of z-average diameters were used. Before analysis, samples were diluted with water to 2% before measurement and measured at 25°C12.



Encapsulation efficiency of cubosome:

Encapsulation efficiency of prepared cubosome was determined using ultracentrifugation. The separation of the free drugs from the entrapped drug in the cubosome dispersion was achieved by centrifugation at 4500 rpm for 20 minute. The supernatant liquid was separated and diluted. The amount of free dug in the dispersion was then analyzed spectrophotometrically at 294nm which was then subtracted from the total amount of drug initially added. The % entrapment efficiency (EE) was calculated by the following equation:12


EE % = (Ctotal con - Cfree con)) / (C total con) × 100%


Determination of Particle morphology by optical microscopy:

Surface morphology of cubosome was determined by optical microscope. An optimized batch of the cubosome was selected. A drop of the cubosome formulation was placed on the glass slide, air dried and covered with a coverslip and observed by optical microscope (Olympus Microscope, Japan) under the magnification of × 10.12


Characterization of cubogel:

Cubogel were evaluated for pH13, drug content12 and viscosity13. pH was determined using digital pH meter (Anamatrix, Mumbai, India). Drug content was determined using UV visible spectrophotometer at 294nm (Shimadzu 1800, Japan). Viscosity of cubogel was measured using Brookfield digital viscometer (model RV-DV2T) at 5-25 rpm using spindle no 4.  


In-vitro mucoadhesion studies of cubogel:

Two in-vitro methods were used to evaluate the mucoadhesive properties of cubogel.


Centrifugation method:

In this method, the mucoadhesive properties of cubogel formulations were evaluated by calculating binding efficiency of mucin with the prepared formulation. The pig mucin was procured from Himedia, Mumbai, India. The mucin suspension (0.1%) was prepared in 0.05 M saline phosphate buffer (pH 7.4). Then 5 ml of cubogel or cubosome formulation was mixed with 5 ml of mucin suspension and the dispersion was incubated at 37 °C±2 °C for 1 hour and kept aside for 24 hour at room temperature. The samples were centrifuged (12,000 rpm) in cooling centrifuge (Remi, R-8C, laboratory centrifuge) for 30 min. Then supernatant liquid was collected and quantified free pig mucin by UV spectrophotometer at 251 nm. Finally, the binding efficiency of mucin with prepared formulation was calculated by following equation:14


% Mucoadhesion=


(Total mucin concentration – Mucin concentration in supernatant)/(Total mucin concentration )  × 100   


Turbidimetric measurements:

Turbidimetric measurements of prepared formulations were compared with mucin dispersion at 251 nm by ultraviolet-visible spectrophotometer. The accurately measured formulation (5 ml) were added to 5 ml aqueous pig mucin dispersion and stirred at 200 rpm. The turbidity of the formulation mucin dispersion was measured at certain time intervals and compared to the turbidity of the mucin dispersion. The increase in turbidity of mucin dispersion indicated mucoadhesive property.15


Statistical analysis:

All experiments were performed in triplicate and data were reported as a mean ±SD. Student's t-test was performed on the data sets using SPSS 16.0 for Windows®. Differences were considered significant for P values <0.05.



Visual inspection:

Cubosome dispersion was observed for physical appearance. Cubosomal dispersion was milky white liquid in appearance (figure 1) and did not show any sign of phase separation. Hence prepared cubosomal dispersion was physically stable.



Figure 1: Timolol maleate cubosome prepared with 6% GMO and 0.6% poloxamer 407


Determination of Particle size, zeta potential and entrapment efficiency:

Particle size of cubosomes has an important effect especially on the dispersion of ophthalmic formulations. Particle size is a vital parameter for absorption or transportation through the ocular barriers and should not be more than 10 μm. In this study, the mean particle size of cubosome was found to be 178.8±8.96 nm and a PDI value of 0.268 (figure 2), showing good uniformity and great potential to transport across the cornea. Surface charge of the cubosome was determined by zeta potential analysis. The zeta potential value was found be -12.2 mV (figure 3). This negative zeta potential was attributed to stabilizing effect of poloxamer 407 adsorbed on the cubosome surface, acting as a coating layer to prevent aggregation. Although the zeta potential in this system was not high enough (>20 mV) to provide effective electric repulsion to avoid the aggregation of particles, Poloxamer 407, a block copolymer containing both hydrophilic (polyethylene oxide) part and hydrophobic (polypropylene oxide) part, would sterically stabilize the cubic phase and preserve the inner colloidal stability of dispersed liquid crystalline particles. The entrapment efficiency of the cubosomes was 98.91± 3.4%, implying that all most all drug was encapsulated in the cubosomes. Results of all parameters are showed in table 2.


Table 2: Results of particle size, PDI, zeta potential and EE of cubosome


Particle size (nm)


Zeta potential (mV)

Entrapment efficiency

EE (%)

Cubosome (A6)







Figure 2: Particle size distribution of cubosome



Figure 3: Particle size distribution of cubosome


Particle morphology by optical microscopy:

Surface morphology of prepared cubosomes was examined under optical microscope. When the samples were visualized using optical microscope, cubosomes were seemed to be bilayer shaped with smooth surfaces. Photomicrograph also showed that cubosomes are well separated from each other. These findings indicated the successful formation of cubosomes (figure 4).


Characterization of cubogel:

pH is one of the most important parameter involved in the ophthalmic formulation. The two areas of critical importance are the effect of pH on solubility and stability. Ophthalmic formulations should have pH range in between 5 to 7.4. The observed pH was lies in the ranges between 6.1-6.7. Drug content was determined using UV method. The drug content was found to be 97.28±0.048% and 98.95±0.041% respectively, for CG1 and CG2 formulation.  This shows uniform distribution of the drug in cubogel formulation. The viscosity of TM-loaded cubogel was compared with cubosome dispersion. The viscosity of cubogel formulation CG1 and CG2 was found to be 169±5.59 cps and 178±6.84 cps respectively at shear stress of 5 rpm, whereas cubosome dispersion showed 78.88±5.22 cps The viscosity of cubosome was significantly increased when formulated in cubosomal dispersion (P<0.05). This might be due to the fact that addition of mucoadhesive polymers (chitosan, gellan gum and carbopol p934) in cubosomal dispersion may be responsible for increased viscosity in case of cubogel.  Results of all parameters are showed in table 3.



Figure 4: Optical microscopy image of cubosome


Table 3: Results of characterization studies of cubogel  



Drug content (%)

Viscosity at 5 rpm (cps)

Cubosome (A6)













In-vitro mucoadhesion studies:

Mucoadhesive strength of prepared formulations was determined by two methods. In first method, mucoadhesive strength was determined by centrifugation method, where cubogel formulation CG1 (94.44±2.180%) and CG2 (91.20±2.590%) showed excellent mucoadhesive strength compared to cubosome (72.79±2.720%).  Results of centrifugation method are showed in figure 5.


In another method, turbidity of drug loaded cubogel-mucin dispersions were examined to obtain information about the mucoadhesive property of prepared formulation. The absorbance was taken up to 8 hours. The absorbance of the mucin-free aqueous cubosomal dispersions of Timolol maleate did not significantly deviated absorbance from zero (0.045-0.057). Therefore, some changes occurred in the turbidity of cubogel–mucin dispersions was considered as an indication for an eventual interaction occurred between mucin and cubogel, and not due to the motion of particles. Cubogel-mucin dispersions showed higher turbidity after 5 hours suggested that the interaction between particles and mucin increases. The turbidity of cubogel-mucin dispersions was higher than that of mucin dispersion alone (figure 6). This phenomenon might be due to the higher thickness of gellan gum (CG1) and chitosan (CG2) around liquid crystalline nano particles (cubosome These findings were in agreement with another study conducted by Yoncheva K et al., 2011 and Kesavan K., et al 2010, where the authors reported higher mucoadhesive property of chitosan and gellan gum respectively in opthalmic formulations.



Figure 5: Comparative mucoadhesive strength of cubogel and cubosome by centrifugation method



Figure 6: Estimation of the interaction between cubogel formulation and 0.1% mucin dispersion by turbidimetric method



Due to the poor residence time and mucoadhesive behaviour of conventional eye drops. There is an urgent requirement for the development of such formulations which not only increases the residence time of opthalmic formulations but also decreases the number of drug instillation. In this study Timolol maleate loaded cubosome was prepared by top down technique and then cubosome was dispersed in in-situ gel in 1:2 ratio to form cubogel. Gellan gum and chitosan were used as polymer and carbopol p934 was used as co-polymer. Cubosomal dispersion was milky white in appearance and having particle size of 181 nm. Viscosity of cubogel was slightly higher than cubosomal dispersion. Mucoadhesive studies showed Timolol maleate cubogel formulation was significantly higher than cubosome. Hence, it can be concluded that cubogel could reduce the problems associated conventional eye drops and improve patient compliance.



We are thankful to Principal, Sri Adichunchanagiri College of Pharmacy, Bangalore, and Rajiv Gandhi University of health sciences, Bangalore, for providing all necessary facilities to carry out this PhD research work.



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Received on 28.07.2018          Accepted on 11.10.2018

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(1): 07-11.

DOI: 10.5958/2231-5691.2019.00002.9