Phase behavior study of Ciprofloxacin hydrochloride Microemulsion prepared by mixed Nonionic surfactant

 

Shashikant Chandrakar *, Amit Roy, Ram Sahu

Columbia Institute of Pharmacy, Raipur (C.G.) 493111 India

 

ABSTRACT:

Ciprofloxacin hydrochloride is water soluble fluoroquinolone derivative antibacterial agent used for bacterial infection in the eye. It is also available in market as eye drop in aqueous solution. The problem with aqueous solution is that it shoes low permeability and requires frequent doing due washing action and blinking reflex in the eye. To overcome this problem microemulsion were prepared. The aim of present study was to develop and evaluates microemulsion of Ciprofloxacin hydrochloride for ocular delivery of drug. Pseudoternaty phase diagram was prepared by using span 80, tween 20 as mixed surfactant, sesame oil as oil phase and double distilled water as aqueous media. The nine microemulsion (M1 to M9) were prepared and evaluated for globule size, zeta potential, polydispersity index, percentage drug content, viscosity, conductivity, percentage transmittance and pH.The globule size of M1 to M9 was in the range 116.6±0.07 to 278.4±0.06 nm. The zeta potential values ranged between +11.23±0.11and +24.56±0.17 mV. The PDI value shows narrow size distribution of globule. The drug content of microemulsion varies from 98.12±0.37% to 99.54± 0.36%.The viscosity value for the microemulsion was found <20 cp. The conductivity values from 0.011±0.18to 0.042±0.62.The percentage transmittance of microemulsion varies from 97.1±0.15% to 99.8±0.11 %. The pH values of microemulsions were found acceptable for ocular application. From the size analyses it was observed that microemulsion M1 to M6 showing narrow size range (<200 nm) and it was selected for further study.

 

 

 

1. INTRODUCTION:

Ciprofloxacin hydrochloride, is fluoroquinolone derivative, is broad spectrum antibacterial agent. It is mostly used for treatment of infection in eye⁽¹⁾. The mechanism of action of ciprofloxacin hydrochloride is due to inhibiting enzymes involved in bacterial DNA synthesis. It causes bacterial cell death. The ciprofloxacin hydrochloride is interacted with pathogenic bacteria in peripheral tissue⁽²⁾. Thus, antimicrobial activity of ciprofloxacin depends on concentration of drug in infected site. Ciprofloxacin hydrochloride is for ocular delivery is available in aqueous solution. There is problem with ophthalmic solution ^(3,4,5) .

 

The bioavailability of drug present in aqueous solution for ophthalmic use is very poor⁽⁶⁾. The reason for that is various constrains present on the eye such as blinking, baseline and reflex lachrymation, and by drainage system present in eye removes drugs, from the surface of the eye.  After instillation of an eye drop, typically less than 5-10 % of an applied dose reaches the intraocular tissues ^(7,8). This is due to restriction caused by corneal barrier and rapid loss of the instilled solution through nasolachrymal drainage. Therefore small amount of drug is available for therapeutic response and it needs frequent dosing of therapeutics agent. The ocular bioavailability of the topical administered drug can be increased by increasing the corneal permeability and by increasing   contact time of drug on the ocular surface.  After the penetration of  the drug through the  cornea or sclera, kinetics of drug transport dependent physicochemical properties of drug like  molecular  weight or molecular volume, and binding  characteristics of drug  tissue binding sites, clearance  of the fluid  reservoirs present in the eye⁽⁹⁾.  This approach of ocular drug delivery can possible when a non – toxic and non irritating permeation enhancer is used in dosage form. This strategy is used in Microemulsion. Microemulsion is composed of surfactant, co-surfactant, oil phase and aqueous phase ^{(10, 11, 12)}. The aim of this study was to formulate microemulsion by using nonionic surfactant, sasame oil and water. The effect of ratio of surfactant, on globule size, PDI, drug content viscosity, pH, conductivity, size etc. were observed.

 

2. MATERIAL AND METHOD:

The nonionic surfactant, span 80, span 20, tween 80, methanol, disodium hydrogen phosphate, potassium dehydrogenate phosphate ethanol, sodium hydroxide, potessium chloride were purchased from Merck India. The Ciprofloxacin Hydrochloride was obtained as gift sample from Sun Pharmaceutical Limited, Dewas (MP). The aqueous media used for preparation of micromeulsion was double distilled water prepared in Lab. The entire chemicals used were analytical grade.

 

2.1 Selection of surfactant

The surfactant was selected on basis of literature survey. The km ratio was determined on the basis of mixing surfactant in different ratio with oil. When the clear phase was obtained such type of ratio was selected for construction of pesudoternary phase diagram ^{(13, 14, 15)}. The compositions of microemulsion were depend on microemulsion area present in ternary phase diagram (Table 1).

 

2.2 Solubility study of Ciprofloxacin Hydrochloride in various excipients

The solubility Ciprofloxacin Hydrochloride in surfactant, co-surfactant was determined. Solubility studies were conducted by placing an excess amount of drug in each 2ml of vehicle. Then the mixture was vortexed and kept for 48hrs at 25ºC in a Orbital shaker (Remi ltd.) to facilitate the solubilization. The samples were centrifuged at 3000rpm for 15min to remove the undisclosed drug. The supernatant was taken and pass though0.45µm membrane filter and the concentration of drug in each vehicle was quantified by U.V-spectrophotometer ^{(14, 16)}.

 

2.3 Construction of pseudoternary phase diagram

For preparation of microemulsion, the concentration the Smix, oil, and water in appropriate ratio is required. Pseudo-ternary phase diagrams were constructed by water titration method at room temepertaure.Soyabean oil was used as oil phase and span80, tween 20 was used as surfactant. Distilled water was used as an aqueous phase. Three phase diagrams were prepared with the 1:1, 1:2 and 1:3weight ratios of Smix, respectively. For each phase diagram at a specific surfactant weight ratio, the ratios of oil to the mixture of surfactant were varied as 1:9 to 9:1; the mixtures of oil, surfactant at certain weight ratios were titrated by adding water drop wise with stirring in magnetic stirrer. When the entire component was mixed properly, the prepared mixtures were visually observed for clear/transparent or translucent mixture. If it was clear and shows flowabilty it is microemulsion. If the prepared microemulsion was highly viscous and it did not show change in height of meniscus when it was tilted it was considered as gel or liquid crystal ^{(16, 17)}.

 

2.4 Preparation of microemulsion

The microemulsion was prepared according to the composition of given in table 2. The drug was dissolved in aqueous media, and then surfactant mixture was added with stirring in magnetic stirrer vat 25⁰C.thw oil was added deopwise in the above mixture. The prepared micoremulsion was further evaluated for different parameter ^{(16, 17)}.

 

2.5 Phase separation study

The small amount of sample of the above prepared microemulsion was taken in centrifugation tube and centrifuged at 5000 rpm for 15 min in cooling centrifuge (Remi India limited) for phase separation study of microemulsion^(17,18).

 

2.6 Globule size, Zeta potential and polydispersity index (PDI) analysis and drug content

The average globule size and PDI of micro emulsions were deter-mined by D LS. Measurements were made using Malvern Zetasizer. The zeta potentail was also measured. It is measured for the stability of microemulsion. Drug content of microemulsions was analyzed by UV–visible spectrophotometer (Shimadzu 1800, Japan) at 275 nm ^{(16,- 18)}.

 

2.7 Conductivity

The electrical conductivity measurements were carried out at 25 ± 0.5 °C using E conductivity meter (Electronic India). The conductivity meter was calibrated using standard KCl solutions. The electrode was dipped into samples and reading became stable then conductivity was measured. The electrical conductivity measurements were conducted in triplicate and the average of the experimental data was use ^(14-18).

 

2.8 pH

The Additive used in the preparation of microemulsion will affect the pH of the preparation .On Literature study it is observed that change in pH may alter the surface charge and hence zeta potential of formulation, which may affect the stability of microemulsion. Therefore, pH is also responsible for stability of micro emulsion. The pH value of ME was determined using digital pH meter (Elico India), standardized using pH 4 and 7 buffers before use ^{(14, 17, 18)}.

 

2.9 Viscosity

The viscosity of mixed microemlusion was measured by using Brookfield viscometer DV-II at 25 ± 0.1 °C. The solution was equilibrated for 5 min to get a constant viscosity value. The measurements of viscosity were conducted in triplicate ^{(14, 16, 17)}.

 

2.10 Statistical Analysis

The values were expressed as mean ± SD.

 

3. RESULT AND DISCUSSION:

Table1: Selection of surfactant and oil phase

Oil	Surfactant  Mixture	KM Ratio	Observation
Sesame oil	Span  20:Tween 20	1:1	Hazy
Sesame oil	Span  20:Tween 20	1:2	Hazy
Sesame oil	Span  20:Tween 20	1:3	Hazy
Sesame oil	Span  80:Tween 20	1:1	Clear
Sesame oil	Span  80:Tween 20	1:2	Clear
Sesame oil	Span  80:Tween 20	1:3	Clear
Sesame oil	Span  20:Tween 80	1:1	Hazy
Sesame oil	Span  20:Tween 80	1:2	Hazy
Sesame oil	Span  20:Tween 80	1:3	Hazy
Sesame oil	Span  80:Tween 80	1:1	Hazy
Sesame oil	Span  80:Tween 80	1:2	Hazy
Sesame oil	Span  80:Tween 80	1:3	Hazy

 

 

Table 2: Composition of Microemulsion

Formulation	Water (%wt)	Oil (% wt)	Smix 1:1 (%wt)	Smix 1:2 (%wt)	Smix 1:3 (%wt)	Drug (% wt)
M1	10	30	60	-	-	0.3
M2	13	27	60	-	-	0.3
M3	16	24	60	-	-	0.3
M4	10	40	-	50	-	0.3
M5	15	35	-	50	-	0.3
M6	20	30	-	50	-	0.3
M7	10	50	-	-	40	0.3
M8	15	45	-	-	40	0.3
M9	20	40	-	-	40	0.3

 

 

3.1 Solubility of Ciprofloxacin Hydrochloride

Solubility of Ciprofloxacin Hydrochloride in surfactant and water are shown in table 3. The water exhibited highest solubility compared to other solvents.

 

 

Table 3: Solubility of ciprofloxacin Hydrochloride in surfactant and water

Solvent/Surfactant	Solubility(mg/g)
Water	30.2±0.03
Phosphate buffer pH (7.4)	28.3± 0.15
Tween 20	8.1 ±0.02
Span 80	2.3 ±0.06

Values reported as mean ± SD; (n = 3)

 

 

 

3.2 Selection of surfactant

The surfactant was selected on the basis of result shown in table 1, miscibility of oil with different ratio of surfactant mixture was considered for selection of surfactant. Span 20 and tween 80 was selected for at ratio of 1:1 to 1:3. When the clear phase was obtained such type of ratio was selected for construction of pesudoternary phase diagram. The result is shown in table 1.

 

3.3 Phase behavior study

For different ratio of surfactant three phase, diagram were constructed comprising different Smix ratio (Span 80: Tween 20) i.e., 1:1, 1:2, 1:3. These pseudo-ternary phase diagrams (consisting oil, Smix and water) demonstrated an extensive region of microemulsion formation (Fig 1 to Fig 3). In addition, phase diagrams also help in determination of concentration range of components used for formulation of microemulsion ^{(18, 19)}.

 

Figure 1: Phase diagram for surfactant mixture (Span 80: tween20) at 1:1

 

 

 

 

Figure 2: Phase diagram for surfactant mixture (Span 80: tween20) at 1:2

 

 

Fig 3: Phase diagram for surfactant mixture (Span 80: tween20) at 1:3

 

 

 

 

 

 

3.4 Globule size, Zeta potential, PDI, and Drug content

Globule size of microemulsions was found in the range 116.6±0.07  to  278.4±0.06 nm (Table 4). It was observed that globule size enhances on increasing the concentration of internal phase. The size reduces on enhancing surfactant concentration. The zeta potential values ranged between +11.23±0.11and +24.56±0.17 mV (Table 4). This variation may be due to diffusion of drug in interface. The zeta potential is determined to predict the physical stability of colloidal systems. Theoretically, the higher the zeta potential value stables the colloidal system. Here the zeta potential value is positive. The polydispersity index (PDI) of the prepared microemulsion was found between 0.189±0.05 to0.360±0.08 (Table 4) which is below 0.4 that is showing narrow size distribution of globules. From the literature it was observed narrow size distribution of globule, PDI ranges from 0.01 to 0.5.The dispersion with broad size distribution have PDI > 0.7^{(19, 20).}.The drug content of microemulsion varies from 98.12±0.37% to 99.54±0.36 % ( Table4).

 

 

Table4: Globule size, Zeta potential, PDI, and Drug content of prepared microemulsion

Formulation	Phase separation	Globule size nm	Zeta Potential mV	PDI	Drug Content
M1	Not Obtained	116.6±0.07	+11.23±0.11	0.189±0.05	99.08±0.26%
M2	Not Obtained	123.3±0.04	+14.21±0.16	0.272±0.02	99.18± 0.31%
M3	Not Obtained	134.1±0.06	+15.14±0.13	0.360±0.08	99.24± 0.46%
M4	Not Obtained	141.7±0.08	+21.34±0.12	0.235±0.03	99.37± 0.22%
M5	Not Obtained	153.2±0.07	+16.26±0.14	0.228±0.09	99.54± 0.36%
M6	Not Obtained	168.4±0.03	+18.48±0.12	0.197±0.03	98.33± 0.49%
M7	Not Obtained	217.2±0.04	+24.56±0.17	0.199±0.04	98.42± 0.37%
M8	Not Obtained	242.1±0.05	+21.15±0.18	0.208±0.05	98.23±0.27%
M9	Not Obtained	278.4±0.06	+23.68±0.16	0.128±0.06	98.12±0.37%

Values reported as mean ± SD; (n = 3)

 

3.5 Viscosity, conductivity, %Transmittance and pH

The viscosity is important parameter to assess the ocular formulation; hence low viscosity causes fast drainage of formulation and reduces retention time. The viscosity value of above prepared microemulsions varied from 11.21±0.16 to 18.21±0.21 cP (Table 5).The above range of viscosity for microemulsion is acceptable for ophthalmic formulation hence eye drops should have viscosity <20 centipoise. The variation in viscosity of prepared microemulsion is due to variation in concentration of surfactant. The M3 revealed higher viscosity due to presence of higher surfactant concentration as well low amount of water. While the M7 prepared with lower surfactant concentration with higher amount of water, and shows low viscosity of microemulsion. The conductivity values of the microemulsions ranged from 0.011±0.18to 0.042±0.62 (Table5).The conductivity values were less than 0.05µS/cm which indicates that the prepared microemulsions were water-in-oil type. The microemulsion prepared by a nonionic surfactant has negligible charge, which resulted in low electrical conductivity ^(21-22).

 

The percentage transmittance of microemulsion varies from 97.1±0.15% to 99.8±0.11 %( Table5).It showed acceptable value for ocular formulation. Low% transmittance value for M1 formulation is due to higher amount of surfactant. As surfactant concentration decreases the clear microemulsion was obtained. The pH values of microemulsions were varied from the range 6.64±0.16to 6.93±0.11 (Table 5). The pH for ophthalmic solutions may range from pH 4.5 - 11.5 but to prevent corneal damage the pH should be 6.6 to 7.8. The eye has limited buffering capacity. the pH values of the prepared microemulsion should be considered as nonirritating to the eye ^{(23, 24)}.

 

 

 

 

Table 5: Viscosity, Conductivity, %Transmittance and pH of prepared microemulsion

Formulation	Viscosity(cp)	Conductivity(µS/cm)	% Transmittance	pH
M1	14.41±0.11	0.011±0.18	97.4±0.16%	6.68±0.11
M2	17.22±0.17	0.016±0.31	97.3±0.11%	6.64±0.16
M3	18.21±0.21	0.019±0.22	97.1±0.15%	6.67±0.21
M4	13.12±0.14	0.021±0.34	98.7±0.16%	6.73±0.12
M5	14.21±0.13	0.023±0.43	98.5±0.14%	6.86±0.14
M6	16.16±0.12	0.028±0.35	98.1±0.15%	6.72±0.19
M7	11.21±0.16	0.029±0.52	99.8±0.11%	6.74±0.14
M8	12.31±0.13	0.034±0.26	99.6±0.16%	6.82±0.23
M9	13.13±0.15	0.042±0.62	99.2±0.13%	6.93±0.11

Values reported as mean ± SD; (n = 3)

 

 

4. CONCLUSION:

Microemulsions of Ciprofloxacin Hydrochloride using nonionic mixed surfactant were developed and characterized for different parameter. All the parameter were acceptable for ocular formulation accept globule size M7 to M9. The globule size of M7 to M9 was greater than 200 nm. Narrow size range microemulsion (<200 nm) are consider better for ocular drug delivery. Further, M1 to M6 will be proposed to carry out ocular irritation, drug release, drug permeation and stability study.

 

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Received on 04.10.2017         Modified on 25.11.2017

Accepted on 21.02.2018      © RJPT All right reserved