Novel ophthalmic timolol meleate liposomal-hydrogel and its improved local glaucomatous therapeutic effect in vivo

Abstract

To overcome the limitations of common eye drops, the study developed a novel timolol mealate (TM) liposomal-hydrogel to enhance drug permeability and prolong residence time in the precorneal region, which achieved more effective local glaucomatous therapeutic effect. Firstly, TM liposome was prepared by an ammonium sulfate gradient-pH regulation method, which its entrapment efficiency reached up to 94% and its averaged particle size is 187 nm with narrow distribution. The corneal permeability through isolated rabbit cornea was measured by modified Franz-type diffusion cells. The results of trans-corneal penetration exhibited that the apparent permeability coefficients (P_app) and the flow rates of steady state (J_ss) of TM liposome was 1.50-fold higher than that of the commercialized eye drop, while TM liposome with 0.02% transcutol P was 2.19 times. In order to increase the retention time and improve the stability of liposome, we further developed a TM liposomal-hydrogel formulation by adding 1.0% HPMC K4M in TM liposome. The results showed an stability during a 120 days storage period than TM liposome. Precorneal retention study in vivo indicated that the optimal liposomal-hydrogel formulation had improved bioavailability and its retention time on rabbit corneal surface were significantly longer than that of pure liposomes or eye-drops. No obvious irritations to rabbit eyes were observed by histopathology microscopy after 7 days exposure.. Comparing to the eye drops, the TM liposomal-gel displayed prolonged therapeutic effect in cornea and greatly lowered the intraocular pressure IOP on the eyes of normal and glaucomatous pigmented rabbits.

Keywords::

• timolol mealate
• ammonium sulfate gradient- pH regulation
• liposome
• liposomal-hydrogel
• ocular delivery
• glaucoma

1. Introduction

The perrequisite of ophthalmic delivery is to obtain and maintain an adequate concentration of drug in the precorneal area. Although approximately 90% of ophthalmic drug formulations in clinics is eye-drops, the eye drops display poor bioavailability and poor patient compliance[1]. Tear turnover and drug binding to tear fluid proteins are additional precorneal factors that contribute to the poor ocular bioavailability of many drugs, when instilled in the eye corna in the form of solution dosage. Liposomes, as valuable and biocompatible drug delivery systems, are widely administered for topical treatments of diseases. Liposomes are suitable carriers of hydrophilic and hydrophobic drugs and can be used to increase drug activity and reduce toxicity. Due to their ability above, they also have a potential for ocular application. However, the major disadvantage in using liposomes lies topically in the liquid nature of the preparation. When getting in contact with the ocular surface, liposomes, as alien bodies, tend to be washed away by reflex tearing. Although positively charged liposomes enhance its adsorption to mucin layer overlying the corneal epithelium, which results in improved drug transfer, they were found to be toxic because of their stearylamine content which can cause pain and unpleasantness after instillation. On the other hand, neutral liposomes were found to be safe for ophthalmic applications [2]. Some hydrogel, e.g. hydroxypropylmethylcellulose (HPMC), is known to be physiologically tolerated for their abundant use as viscosity enhancer in aqueous eye drops [3]. It has been reported that the HPMC K4M has good bio-adhesive and hydrophilic properties for eyes [4]. Liposomes exclusively stabilized with HPMC are thus interesting dosage forms for ophthalmic application. Due to the good bio-adhesive and hydrophilic properties of HPMC, the residence time of liposomes in the precorneal region gets prolonged.

In our studies, TM, as a water soluble model drug, is one of the drugs at choice for treatment of open angle glaucoma [5], by inhibiting the production of aqueous humor and lowering the pressure inside the eye. A number of side effects, such as heart failure, can be caused simply by its ophthalmic use[6]. Therefore, a liposome system would be a better choice for this kind of salt of timolol and could obtain optimal corneal penetration of TM. Moreover, considering the stability and pharmacodynamics, liposomal TM was further incorporated into a bio-adhensive HPMC in our study in order to reach superordinary storage stability of formulation and prolonged local therapeutic effect on glaucomatous disease with limited systemic absorption and side effects.

2. Materials and methods

2.1. Materials

Phosphatidyl-choline (PC, soybean lecithin) and Cholesterol were kindly gifted by Lipoid (Germany) and Country medicine group Shanghai Company (ShangHai, China) respectively; Timolol mealate was purchased from SuZhou No.1 Pharmaceutical Manufacture (SuZhou, China); HPMC K4M was kindly gifted by Colorcon (UK); Trancutol P were kindly provided by Gattefosse; All other reagents were of analytical grade.

2.2. Preparation of TM liposomes (TL) and liposomes with Trancutol P (TLP)

To deliver a sufficient amount of drug providing therapeutic effect, a high trapping efficiency of drug in liposome is required. One of the limits connected with almost all preparation methods is the poor entrapment of water-soluble compounds into liposomes [7]. Therefore, based on the properties of TM, a ammonium sulfate gradient-pH regulation method was adopted in our study to prepare drug liposomes.

Firstly, liposomes without TM were prepared using the thin-film dispersion method [8]. The required amount of lipids (the weight ratio of PC and cholesterol is 5:1)were dissolved in 10ml of chloroform in a round-bottom flask. Then, the organic solvent was slowly removed using rotary evaporator (RE -85Z, Gongyi, China) at 40 °C such that a very thin film of lipids was formed on the inner surface of the flask. The flask was left under vacuum for about 12 h to ensure the evaporation of all traces of chloroform. The dry lipid film was slowly hydrated with 10 ml of 250 mM ammonium sulfate solution. The liposomal suspension was mechanically stirred at 40 °C to produce large multi-lamella vesicles (LMV).The LMV was then treated by probe-type ultrasonic for 3 min (active every 5 s for a 5 s duration, 400 W) under the condition of ice bath. Unentrapped ammonium sulfate was removed from the liposomal suspension by dialysis against isotonic 0.9% (w/v) NaCl for about 24 h. In order to establish an optimal trans-membrane pH gradient, the liposomal suspension was titrated to various pH values ranging from 6.0 to 10.0 by using 0.1N NaOH. The pH value of the liposomal suspension was verified after the addition of drug. Timolol maleate was The suspension was incubated at 40 °C for 30 min with intermittent stirring and the TL solution was finally obtained after removing the residual TM from the liposome formulation by dialysis against isotonic 0.9 % (w/v) NaCl.

Additionally, the TLP solution was formulated by adding 0.02% (w/v) of Trancutol P, as an penetration enhancers, into the liposomal suspension, based on the same preparation method for TL.

2.3. Preparation of TLP hydrogels (TLPG)

The different amount of HPMC K4M solid powder was weighted and added slowly into the liposomal suspension with the aid of magnetic stirring. After addition of the full amount of solid material, the gels were allowed to swell under moderate stirring for at least 24 h until fully swollen. For gel preservation, sodium benzoate (0.02%, w/v) was added in the liposomal suspension used for gel preparation [9].

2.4. Determination of entrapment efficiency

The entrapment efficiency (E%) was measured by gel permeation chromatography [10] on a Sephadex G-50 column (15mm×200 mm). An appropriate amount of liposome sample was loaded onto the gel column and eluted continuously with isotonic 0.9% NaCl at a speed of 1.0 ml/min. The eluants were collected separately in numbered tubes to separate entrapped drug from free drug, and were diluated by 5 times the amount of alcohol. The absorbance of the diluted solutions were measured at 296 nm with a UV spectrophotometer (UV-9100, Shanghai, China). Each determination was run in triplicate. The E% was calculated according to the following equation:

1

where E (%) is the trapping efficiency, ED is the concentration of total amount of trapping drug, and UED is the concentration of free drug.

2.5. Particle size and zeta potential measurement

The mean particle size and size distribution of the liposomal dispersion were measured by a laser particle size analyzer (LS230, Beckman-Coulter, USA), and the zeta potential was measured by a zeta potential analyzer (Delsa 440SX, Beckman-Coulter, USA).

2.6. Transmission electron microscopy

A small aliquot of the liposomal dispersion was negatively stained using phosphotungstic acid (PTA, 1%) and the samples were examined using a electron microscope (JEM-1200EX, Tokyo Japan) employing an accelerating voltage of 60 kv.

2.7. Rheological characteristic of TLPG

Viscosity of TLPG containing different amount of HPMC K4M (0.5%, 1.0%, 1.5%, 2.0%, w/v) were determined using a rotating cylinder viscometer (NDJ-8S, Shanghai, China). To test the viscosity of the samples, angular velocity of the rotor was adjusted gradually with No.3 rotator. The samples were equilibrated at 34 °C prior to each measurement. All measurements were made in triplicate and their mean value was reported.

2.8. Stability of TLPG

The storage stability of TLPG formulation was investigated as follows. Briefly, TLPG formulations sealed in glass bottles were stored at 4 and 25°C and observed visually as well as for microscopic appearance at the end of 15, 30, 45, 60, 90 and 120 days from the date of preparation [11].

2.9. In vitro trans-corneal permeation studies

Male New Zealand white rabbits (male, weighing 2.5–3.0 kg) were used. All animal studies were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication No. 92–93, revised in 1985), and were approved and performed in accordance with the guidelines of the institutional Animal Ethics Committee. After the rabbits were sacrificed by intravenous air injection. The cornea was excised immediately, weighed and preserved in glutathione bicarbonate Ringer (GBR) buffer [12]. The corneal permeation studies were carried out using the modified Franz-type cells at 34°C. The corneal was sandwiched between donor and receptor. The volume of GBR buffer filled into the receptor chamber was 7.8 mL and 1ml of liposomal suspension (TL or TLP contaning 0.34% TM) was applied. In reference cell, 1ml of TM eye drops (TD) (containing 0.34% drug in 0.9% NaCl) was used as control. The corneas should be installed into the Franz-type cells within 20 min after excision, and the available area for diffusion was 0.70 cm². At time intervals of 30, 60, 90, 120, 180 and 240 min, 1 ml of sample was withdrawn from the receptor chamber and an equal amount of GBR buffer was added to maintain the original volume. Each experiment was run in triplicate. The concentration of the drug in samples was determined by HPLC. The HPLC system was composed of a model LC-20AT pump (Shimadzhu, Tokyo, Japan) and a model SPD-20A UV detector (shimadzhu, tokyo, Japan). A Diamasil C18 column (200 mm×4.6 mm, 5 μm, Dikma, China) was used. The mobile phase was a mixture of methanol, water and triethylammonium (50:50:0.1, pH 3.5 adjusted by phosphoric). The flow rate was 1.0 ml·min⁻¹, the detection wavelength was 296 nm, the injection volume was 20 μL and the column temperature was 25 °C.

The apparent permeability coefficient (P_app) and the flow rate of steady state (J_ss) were calculated as follow [13]:

2 3

where the term ΔQ/Δt is the steady-state of the linear portion of the plot of the amount of drug in the receptor chamber vs time, A is the available cornea area for diffusion (0.70 cm²), C₀ is the initial concentration of drug in the donor cell and 60 is the conversion of units from minute to second.

2.10. Corneal hydration levels measurement

The corneal hydration level (H%)was calculated from Eq. (4) [14]:

4

where W_b is the wet cornea weight, and W_a is the corresponding dry cornea weight after a desiccation of 12 h at 70 ^°C. The corneal hydration levels were mearsured for both newly excised corneas (no later than 20 min after removal from the rabbit eyes) and treated corneas (after the corneal permeation tests)[15].

2.11. In vivo precorneal retention studies

In vivo precorneal retention of TD, TLP and TLPG on rabbits cornea were determined by the following method [15]. The rabbits were divided into three groups, with each group composed of three rabbits (six corneas). After instilling 50 μl sample into the eyes of rabbits, at time intervals of 5, 15, 30, 60, 120, 180 and 240 min, a little piece of filter paper (2 mm×5 mm) was put into the conjunctival sac and remain for about 5 s, then a 1.5 mL tube was placed immediately, the total weight was determined before and after the tear being adsorbed (the volume of tear could be calculated by the difference of the weight). Then 500μl of methanol was added into the tube, ultrasonic for 15 min, take out of the filter paper and blow to dry through nitrogen. Subsequently the strips were added 500 μL of methanol and then was extracted by sonication for 15 min in a bath type sonicator (KQ-100DB, China) followed by vortex for 3min. The extract fluid was filtered by 0.45 μm membrane and dried under a nitrogen flow and stored at -18 °C. The samples were re-dissolved with 200 μL of methanol before HPLC analysis. The HPLC conditions were as described in Section 2.9. Pharmacokinetic parameters were calculated by statistical moment method [16].

2.12. In vivo ocular irritation studies

Twelve New Zealand white rabbits (male, weighing 2.5-3.0 kg) were used to evaluate the ocular tolerance of the preparations (TD, TLG and TLPG), and divided into four groups with untreated rabbits used as control. For each single instillation, 50 μl of samples were instilled into the lower conjunctival sac. For acute irritation, the rabbits received 3 consecutive instillations with 10 min intervals, and 30 min after the treatment the rabbits were examined for signs of ocular irritation. For long term irritation, the rabbits received instillations 5 times a day for 7 days, and the rabbits were examined at the end of the treatment. The irritation level was evaluated by the animal discomfort and symptoms and signs in the conjunctiva, cornea, and lids (Table 1), according to the scoring system of guidelines for ocular irritation testing [17]. After the examination, the rabbits were euthanized by air embolism, and the eye tissues (cornea, conjunctiva, iris and sclera) were fixed by 4% formaldehyde, embedded in paraffin and made into histological section for histopathology microscopy.

Table 1.  Grading of ocular irritation test.

Grade	Disco mfort	Cornea	Conjunctiva	Discharge	Lids
0	No reaction	No alterations	No alterations	No discharge	No swelling
1	Blinking	Mild opacity	Mild hyperemia, mild edema	Mild discharge without moistened hair	Mild swelling
2	Enhanced blinking, intense tearing; vocalizations	Intense opacity	Intense hyperemia; intense ederna; hemorrhage	Intense discharge with moistened hair	Obvious swelling

2.13. Pharmacodynamics

Although rabbits` eyes are physiologically different from those of humans in terms of eyelids, blinking and tear turnover. Some researches [18, 19] have observed that timolol disposition in tear fluid of rabbits provides a good estimation of the behavior in humans.

A total of twelve New Zealand white rabbits were used and divided into two groups, whose initial intraocular pressures (IOP) were measured by using a standardized YZ7A tonometer [20]. All intraocular pressure measurements were carried out after local anesthetized using amethocaine (1%) eye drops. Each group was designated to receive TD and TLPG respectively. Both preparations contained 0.34% TM equivalent to 0.25% timolol by weight.

2.13.1. The functions for normal rabbits

Two groups of rabbits received a single 50 μl dose of corresponding samples, described as above, and then were measured IOP at every hour until the IOP returned to the initial value for each rabbits[21]. Every eye was tested three times to seek average value.

2.13.2. The functions for glaucomatous pigmented rabbits

After instilling 50 μl of samples into eyes of rabbits for about 1 h, 5% glucose solution (15 ml/kg) was injected into the marginal ear vein immediately for causing a quickly rise in intraocular pressure but no apparent macroscopic or microscopic damage to the eye [22]. The values of IOP were measured in administered eyes at 5–10 min, and then every ten minutes until the IOP returned to the initial value for each rabbit. Every eye was tested three times to seek average value. Repeated the experiment and compared the pharmacodynamic difference of two preparations.

3. Results and discussions

3.1. liposome formulation and characterization

Ammonium sulfate gradient-pH regulation method is an appropriate method for preparing water-soluble drug liposome. Fig. 1 showed that the entrapment efficiency (E%) of TM was dependent on the pH value of the external medium. It was obvious that the fraction of entrapped TM nearly doubled as the external pH was increased from 6.0 to 10.0. The highest E% achieved 95% when the external pH was adjusted to 9.2, which was very close to the isoelectric pH of TM (pK_a was 9.21)[23]. However, further increasing the pH value, no increase of E% was found. The reason for that would be the relationship between the E% of drug and its tendency to be accommodated in the liposomal membrane. Generally, entrapment is more easily achieved with lipophilic and amphiphilic molecules [24]. TM presented as ionization form when pH was lower than pKa, while as molecules form when pH was increased to pKa. In brief, following the solubility decreasing and liposolubility increasing, the potention of transfer lip-membrane and entrapment efficiency of TM would increase constantly. The particle size distribution (mean diameter and polydispersity index) of the optimized formulation was presented in Fig. 2. As anticipated, the size of liposomes are small and uniform after being treated with ultrasonic sound with mean size value of 187nm, while d₁₀, d₅₀ and d₉₀ were 155 nm, 186 nm, and 222 nm respectively. The zeta-potential was showed in Fig. 3, and no surface charge was present for thesd vesicles since the values are practically zero (when SD of each measurement is considered). This result was expected since the lipids used for their preparation are uncharged.

Figure 1.  Effect of pH of external loading medium on the E % of TL. TL composed of PC-Cholesterol (weight ratio of 5:1) were prepared by ammonium sulfate gradients- pH regulation methods. Each value represents the mean ±SD (error bars) (n = 3).

Figure 2.  Particle size distribution of TL formulations.

Figure 3.  The zeta potential distribution of TL formulations.

From Fig. 4 (a), it could be seen that the obtained optimal liposomes were mostly spherical and uniform. Fig. 4 (b) showed a typical morphology picture of a single particle. From the micrograph, it revealed that the structure of liposome is a large unilamellar vesicle (LUVs) with a large internal aqueous space, which may be responsible for its entrapment efficiency of TM.

Figure 4.  Transmission electron microscope of TL formulations prepared by an ammonium sulfate gradients- pH regulation method.

3.2. liposomal-Hydrogel formulation and characterization

In our study, HPMC K4M was selected as the vehicle for incorporation of neutral liposomes destined for ophthalmic delivery with prolonged drug action in cornea. Viscosity values of the TLPG containing different amount HPMC K4M (0.5%, 1.0%, 1.5% and 2.0%) were listed in Table 2. The results suggested that the viscosity of formualtion depends on the concentration of the HPMC K4M, and 1.0% seemed to be the optimal concentration since it meets the suitable viscosity range of 4.0-5.0cPa. s for ocular preparation [25].

Table 2.  The viscosity of TLPG containing different concentration of HPMC K4M at 34 °C. All measurements were kept No.3 rotator constant and made in triplicate to seek average value (n = 3).

HPMC K4M concentration	rpm	viscocity(cPas)
0.5%	12	2.92 ± 0.32
1.0%	60	4.87 ± 0.65
1.5%	60	7.07 ± 0.21
2.0%	60	15.99 ± 0.72

The poor physical stability of TM liposomes was apparently appeared after 7 days from the date of preparation at room temperature by our visual observation, which generally due to the sedimentation and aggregation during storage. However, in the case of TLPG, no perceptible change in appearance was observed when stored at room temperature for 30 days and at 4 °C for 120 days from the date of preparation by microscope observation, meanwhile no significant changes were found in pH, viscosity, E% or drug content value (data not shown). This could be due to the physical process during TLPG formualting. In the present study, HPMC powders were mixed into the TLP suspension to form a hydrogel, which not only preserve the original structure of liposomes but also contribute to the favorable dispersion of liposomes.

3.3. In vitro trans-corneal permeation studies

The rabbits were divided into two groups and each group composed of three rabbits. The corneal penetration profiles were shown in Fig. 5, and the corresponding administration regimen, P_app (R²), J_ss, lag time and corneal hydration values were showed in Table 3 (where the linear regression was carried out at the interval of 90–240 min). The penetration profiles were linear in all cases (R² > 0.996), which indicated that the cornea integrity was maintained under the experimental conditions and the penetration rate was constant. For the group 1, TL solution showed a higher corneal permeability (about 1.50 fold) than TD in a period of 240 min. For the group 2, liposomes with 0.02% transcutol P (TLP) significantly increased corneal permeation (about 1.50 fold) compared with the formulation without it. As a surfactant, the action mechanism of Transcutol P on drug corneal transport may involve changes in the structure of the epithelium as a result of its producing micelles in the epithelial lipid bilayer. The micelles formed by Transcutol P result in the removal of phospholipids from the epithelial cell membranes, thereby leading to an increase in the transcorneal passage of drugs.

Table 3.  Transcornealparameters of the preparations in vitro (n = 3, ± SD). For each rabbit, the cornea of left eye (L) was used for reference preparation, while the cornea of the right eye(R) was for test preparation.

Cornea		Preparation	Papp×10^−6/cm s^−1(R^2)	Jss×10^3/μg s^−1crn^−2	Lag time (min)	H%
Group 1	L	TD	1.27 ± 0.18 (0.996)	31.86 ± 2.05	30.61 ± 6.29	80.15 ± 0.87
	R	TL-1	1.91 ± 0.16 (0.999)	47.44 ± 3.32	28.49 ± 5.06	81.04 ± 1.41
Group 2	L	TL-2	1.86 ± 0.13 (0.997)	43.01 ± 1.69	24.72 ± 3.94	80.28 ± 1.46
	R	TLP	2.79 ± 0.22 (0.999)	69.87 ± 4.87	21.83 ± 4.13	79.01 ± 1.69

Figure 5.  Corneal penetration profiles of drug in different vehicles. Each value represents the mean ±SD (error bars) (n = 3).

Hydration levels of cornea were used to evaluate the corneal damage of substances in vitro. The hydration level of healthy cornea is 76–80% [26], and when a hydration level higher than 83% is detected, the cornea is considered to suffer a certain degree of injury. The hydration levels of the excised cornea exposed to three preparations presented satisfactory for ocular use.

3.4. In vivo precorneal retention studies and Pharmacodynamics studies

As shown in Fig. 6, it can obviously found that TLPG system notably prolonged pre-ocular retention time of drug compared with the TL suspension and TD solution, which should be owing to the hydrophilic bioadhesive characteristic of HPMC polymer. At the same time, the drug concentration on the precorneal for TLPG was remained much higher than that for TLP and TD during the initial 30 minutes after administration. The parameters describing the precorneal drainage were summarized in Table 4, including C_15min (drug concentration remaining on the corneal surface at 15min), AUC_5-240min (area under the curve of the drug concentration remaining in the precorneal region versus time), T_1/2 (half-life of drug elimination on the corneal surface), K_e (the rate constant of drug elimination on the corneal surface) and MRT (mean residence time of the drug on the corneal surface). According to the AUC_5-240min values, the bioavailability of drug on the rabbit eyes for TLPG formulation soared significantly (p<0.05), compared with TLP and TD. More precisely, 1.37-fold and 1.61-fold improvement were achieved compared with TLP and TD, respectively, because of the bioadhesive characteristic of hydrogel on surface of rabbits’ eyes. The longest T_1/2 or MRT was achieved by TLPG than either TLP or TD (p<0.05). Based on this phenomenon, it could be predicted that the TLPG can play a more effective treatment on local glaucomatous disease.

Table 4.  Precorneal clearance parameters.

Formulation	C15min( μg min^−1)	AUC5-24 min	Ke (×10^3 min^−1)	T1/2 (min)	MRT (min)
TD	99.094 ± 10.5	17152.64 ± 374.51	3.21 ± 0.18	216.18 ± 12.54	313.937 ± 32.08
TLP	153.074 ± 19.1	20189.84 ± 551.76	2.69 ± 0.21	258.40 ± 20.09	368.67 ± 53.17
TLPG	302.288 ± 33.4	27583.66 ± 966.24	1.64 ± 0.09	423.47 ± 23.18	503.072 ± 75.22

Figure 6.  Drug concentration in precorneal regions following topical instillation. Each value represents the mean ±SD (error bars) (n = 6).

Fig. 8 shows the measured decrease in the IOP of normal rabbit as a function of time after administration with TD and TLPG. Administration with TLPG decreases the IOP from an initial value of 2.21 to 1.59 KPa during a period of 1 h. The lowered IOP remains for 6 h before rises to its initial value. Administration with control drops shows a similar behavior, where the IOP decreases (from 2.21 to 1.95 KPa) and remains at its lower value for remarkably shorter periods of time (p<0.05).

Figure 7.  Histopathology microscopy of the ocular tissues after treated with TLPG for 7 days. A–B: cornea of non-treated (A) and treated with TLPG (B); C–D:conjunctiva of non-treated (C) and treated with TLPG(D).

Figure 8.  The change in the IOP as a function of time for normal rabbits. Every eye was tested three times and to seek average value. Each value represents the mean standard error(error bars) (n = 6).

The same protocols were used for two sets of glaucomatous rabbits and the results were showed in Fig. 9. TLPG showed an improved inhibition function to IOP heighten compared with TD at each time point.The average values of IOP for TLPG group (2.33 KPa) was significantly lower than that for TD group (2.61 KPa) in the period of 60 min (p<0.05). The higher drug concentration and longer retention time for the TLPG formulation, observed in rabbit precorneal retention studies, provide a good explanation for this stronger and prolonged local therapeutic effect on glaucomatous rabbits.

Figure 9.  The change in IOP as a function of time for glaucomatous pigmented rabbits.Every eye was tested three times and to seek average value. Each value represents the mean standard error(error bars) (n = 6).

3.5. Ocular irritation studies

The results of irritation (Table 5) indicated that no signs of discomfort appeared during either acute or long term test, in all the experimental rabbits. The result of ocular irritation test presented satisfactory correlation with the hydration levels of the excised cornea (Table 3). For the symptoms of cornea, conjunctiva and eyelids, only grade 0 (no signs of discomfort) was recorded for both treated and control groups, in either acute or long term test.

Table 5.  The score of ocular irritation test (n = 6).

Formulation	Cornea	Iris	C o nj unc tiva	Total score
TD	0	0	0	0
TLP	0	0	0	0
TLPG	0	0	0	0

Fig. 7 showed the histopathology of the tested rabbit eyeballs after long term irritation test. Normal and healthy structures of ocular tissues were observed in all the tested eyes and there were no differences between TLPG treated groups and control group. Corneal and conjunctival epithelial cells maintained normal morphology and constructed integrated epithelium (Fig. 7A–D). The basal cells of cornea remained abundant and were normally packed by junction complex (Fig. 7A–B). Conjunctival lymphoid tissue was identified in all the conjunctivas without the abnormality of its size and location (Fig. 4C–D). The histopathology confirmed that no ocular irritating effects were induced by TLPG compared with non-treated eyes. The combination of TLGP, both of which are biocompatible, demonstrated a preferable ocular tolerance.

4. Conclusions

In this study, a novel Timolol meleate (TM) liposomal-hydrogel system was successfully developed for achieving a better local therapeutic effect on golaucomatous disease than conventional eye drops. In this system, liposome was used as a carrier for our water soluble drug, and the significant enhancement of drug corneal permeability was achieved by addition of transcutol P (0.02 %) as permeability enhancer. Using HPMC K4M (1.0%) as a gelling agent of liposomes, the storage stability and precorneal retention of formulations were improved obviously. The results of rheological and ocular irritation studies indicated that the hydrophilic polymer of HPMC, acting as a vehicle for incorporation of TM liposomes, was biocompatibility for ocular transmit system. Pharmacodynamics studies suggested that this novel TM loaded liposomal-hydrogel would be a viable alternative to conventional eye drops by virtue of its ability to enhance drug action. Also important is its potential of reducing the frequency of administration resulting in better patient acceptance. Moreover, it is encouraging that this system could have a very high drug loading, which may broaden its applications in other areas such as transdermal drug delivery, and also for delivery of hydrophobic molecules to eyes.

Acknowledgements

This research was financially supported by National Natural Science Foundation of P.R. China (No.30500638). We greatly thank Dr. Ning Li and Dr. Xiang Li for their excellent technical assistance.

Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the article.