INTRODUCTION:

The aim of novel drug delivery system is to provide a therapeutic amount of drug to the appropriate site in the body to accomplish promptly and then maintain the desired drug concentration. The drug-delivery system should deliver drug at a control rate according to necessity of the body over a specified term of treatment¹.Efficacy of a drug can be altered by the method of drug delivery into the body.

Some drugs have an optimum concentration in the body and produce maximum therapeutic level, concentration above or below can produce toxic or no therapeutic action. Slow efficacy of drugs in severe diseases have increase the need for a multidisciplinary approach to the delivery of therapeutics to targets in tissues. To solve this problem newer development of pharmaceutical compounds were generated to control the pharmacokinetics, pharmacodynamics, nonspecific toxicity, immunogenicity, bio recognition, and efficacy of drugs. The drug delivery systems (DDS) which are based on interdisciplinary approaches that combine polymer science, pharmaceutics, bioconjugate chemistry and molecular biology are called as Novel Drug Delivery Systems (NDDS)².

Nanosponges are nanosized free flowing powder particles of approximately size ranging from (250 nm-1 µm)³. They can encapsulate wide varieties of drug molecules, which have the capability of increasing solubility of low soluble drugs (Class-II, Class-IV). Nanosponges also increase bioavailability of the drugs, decrease frequency of dosing as well as reduce various side effects associated with particular API⁴. Nanosponges swell when incorporated in gel base for topical preparations, so these swelled structures get retained on the surface of stratum corneum providing control action at that particular site.

Sertaconazole Nitrate is a highly lipophilic antifungal drug that belongs to the imidazole class which exhibits broad spectrum antifungal activity against dermatophytes and many pathogenic fungi by inhibiting ergosterol synthesis in fungi⁵. Ergosterol is the main component in synthesizing fungal cell membrane, so inhibiting ergosterol synthesis leads to disruption in fungal cell membrane and cause leakage of cell contents. It is a BCS Class-II drug (Low soluble and highly permeable).

The objective of present research work was to formulate sertaconazole nitrate loaded nanosponges and incorporation of these prepared nanosponges in a carbopol gel base. As, selected drug has high permeability and low solubility, to combat these features nanosponges are choosen which can increase solubility and improve skin retention of prepared formulation.

MATERIALS AND METHODS:

Materials:

Sertaconazole nitrate was obtained as a gift sample from varahi Pvt. Ltd., Ethyl cellulose from balaji drugs, Polymethylmathacrylate from research-lab fine chem industries, Polyvinyl alcohol from NR chem, dichloromethane from S.D fime chemicals Ltd., Methyl paraben from S.D fine chemicals Ltd., and Methanol from merck life sciences Pvt. Ltd.

Experimental methodology:

Analytical methodology of sertaconazole nitrate

Absorption maxima for sertaconazole nitrate in pH 7.4 Phosphate buffer saline is determined by using 60µg/ml standard solution was scanned on a double beam spectrophotometer against pH 7.4 phosphate buffer saline as a blank. Sertaconazole nitrate standard curve was performed in pH 7.4 phosphate buffer saline (I.P.) and prepared in the concentrations of 10µg/ml to 60µg/ml and analyzed the samples by UV spectrophotometer^6,7.

Drug excipient compatibility studies:

FTIR study was carried out to check compatibility of drug and excipients. The spectrum analysis of sertaconazole nitrate, polymers, surfactant which were employed in the preparation of nanosponges were studied by Fourier Transform Infra-Red (FTIR) Spectroscopy. FTIR spectra were recorded by preparing potassium bromide (KBr) disks using a Shimadzu Corporation (Kyoto, Japan) facility (model - 8400S). Potassium bromide (KBr) pellets were prepared by mixing few mg of sample with potassium bromide and compacting in a hydrostatic press under vacuum at 6-8 tons pressure. The resultant disc was taken carefully in order to obtain an intact pellet which was mounted in a sample holder in IR spectrophotometer and the IR spectrum was recorded from 4000 cm^-1 to 200 cm^-1. The resultant spectrum was compared for any spectral changes. The obtained spectra was observed for the presence of any characteristic peaks for the respective functional group in the compound⁸.

Preparation of nanosponges:

Nanosponges can be prepared by different methods like quasi emulsion solvent diffusion method, ultra sound assisted method, solvent method and hyper crosslinked cyclodextrin method. Among the four methods quasi emulsion solvent diffusion method is most commonly used in the preparation of nanosponges for topical application due to its simplicity and negligible solvent toxicity. So, nanosponges are prepared by quasi emulsion solvent diffusion method in present work.

Method of preparation:

Sertaconazole nitrate nanosponges were prepared by emulsion solvent diffusion method. Polyvinyl alcohol dissolved in distilled water at 80ºc for 30min is used as aqueous phase. The drug and polymer dissolved in dichloromethane (crosslinking agent) is used as organic phase. Organic phase is subjected to sonication for 10 min in order to increase the solubility of drug in polymer solution. The organic phase is slowly added to aqueous phase to avoid foaming by using fine syringe with a size range of 0.001. This was performed on a Remi magnetic stirrer at 1000rmp for 3hrs at 35ºc. The formed solid powder was collected by using membrane filter (pore size 0.45μm) and dried in oven for 30min at 40°C for the removal of residual solvent. The product was then packed and stored in airtight vials for characterization⁹.

Figure 1: Preparation of nanosponges by magnetic stirrer

Preliminary trails for the formation of nanosponges:

Nanosponges were prepared by emulsion solvent diffusion method by using different polymers at varying surfactant concentration. The five different polymers which were used are ethyl cellulose, polymethyl methacrylate, pluronic F-68, eudragit RL-100 and eudragit RS-100^10,11. The polymers with which nanosponges were formed are selected for further studies.

Determination of entrapment efficiency with different ratios of polymers to optimize drug and polymer ratio:

Different sources have reported different optimized drug and polymer ratios for nanosponges. Inorder, to fix appropriate drug and polymer ratio for sertaconazole nitrate loaded nanosponges, % Entrapment efficiency was calculated with two polymers (ethyl cellulose and polymethyl methacrylate) in six different ratio. The surfactant concentration of 0.2% was used for ethyl cellulose and 0.3% for polymethyl methacrylate. Crosslinking agent (DCM) and solvent (water) were constant⁹. Entrapment efficiency was determined for all the 12 formulations and the best entrapped formulation ratio is chosen for both the polymers.

Optimization of sertaconazole nitrate loaded nanosponge gel using Taguchi OA L⁸ as design of experiment (DOE):

Taguchi OA L⁸experimental design was used to study the effect of different polymers like ethyl cellulose (EC), polymethyl methacrylate (PMMA) at two levels, effect of surfactant like polyvinyl alcohol at two levels of 0.2% and 0.3% and effect of crosslinking agent like dichloromethane at two levels of 10ml and 20ml. Three factors (independent variables) such as type of polymers, percentage of surfactant and amount of crosslinking agent, were studied at all two levels. Entrapment efficiency was taken as the response (dependent variables). An orthogonal array was used for choosing the best and optimized formulation. The resultant formulations were optimized depending on their %Entrapment efficiency

Design of experiment for choosing the best and optimized formulation:

Table-1 indicates the experimental runs to be conducted varying three factors at two different levels.

Table 1: Taguchi orthogonal array (2³) design of experiment

Independent variables	Level A	Level B
Factor A (type of polymer)	Ethyl cellulose	Polymethyl methacrylate
Factor B (Percentage of surfactant)	0.2%	0.3%
Factor C (Amount of crosslinking agent)	10ml	20ml

Table 2: Taguchi orthogonal array (2³) design experimental trials

Trial	Factor A	Factor B	Factor C
1	EC	0.2	10
2	EC	0.2	20
3	EC	0.3	10
4	EC	0.3	20
5	PMMA	0.2	10
6	PMMA	0.2	20
7	PMMA	0.3	10
8	PMMA	0.3	20

Table:3, indicates the formulae for each experimental run. In the above formulae, three factors at two levels were varied while the amount of solvent (water) is kept constant in all the runs. Type of polymers, effect of surfactant and crosslinking agent were studied for all the above eight runs.

EVALUATION OF NANOSPONGES:

a) SEM studies:

Nanosponges can be easily visualized by scanning electron microscopy (SEM). SEM is a powerful microscope that uses electrons to form an image. It allows for imaging of samples at magnifications which cannot be achieved using traditional microscope. Typical SEM can reach magnifications of more than 30,000X, where as modern light microscopes can achieve a magnification of ~1,000X. The resulting pictures it forms are in black and white as SEM doesn’t use light to create images.

b) Size distribution studies:

Particle size of the nanosponge can be determined by dynamic light scattering (DLS). It can be determined by measuring the random changes in the intensity of light which are scattered from a sample. Small particles in the sample undergo random thermal motion called Brownian motion. Light from the laser source illuminates the sample in the cell. The scattered light signal is collected with one of two detectors, either at a 90-degree (right angle) or 173-degree (back angle) scattering angle. The obtained optical signal shows random changes due to random changing position of the particles.

c) Zeta potential:

Zeta potential of the formulation was measured by Zetasizer. Electrophoretic light scattering method was utilized for zeta potential measurement. A dip cell (zen1002, Malvern Instruments) with a pair of parallel Pd electrodes was used to provide electrical trigger on charged particles. The signals were collected at 12.8º and the data were analyzed using Zetasizer Software.

d) Drug entrapment:

Nanosponge formulation of equivalent amount was mixed with 10ml of pH 7.4 phosphate buffer and transferred into an Eppendorf tubes. The obtained solution was centrifuged at 9000rpm at 4ºC for 30 min (two cycles) in order to separate nanosponge loaded drug from free drug. The resulted clear solution was filtered through Whatsmann filter paper and subjected to U.V. which is used for the determination of un-entrapped drug.

% Entrapment efficiency = Amount of entrapped drug /Amount of total drug added * 100

e) Production yield (%):

For calculating production yield, the theoretical mass was calculated initially by taking the mass of solid ingredients added. Prepared nanosponge formulation was accurately weighed and the weight was recorded. The production yield of the nanosponges was then determined using the following equation¹³.

Production yield (%) = Practical mass of nanosponges/Theoretical mass (polymer + drug)*100

Incorporation of optimized formulation into suitable gel

As, nanosponges have low viscosity they should be incorporated in a suitable gel base for ease of application¹⁴. The optimized formulation was incorporated into carbopol gel base. Accurately weighed quantities of Carbopol 934 P was soaked in distilled water for 24 hours, propylene glycol was added slowly with stirring. Accurately weighed sertaconazole nitrate loaded nanosponges were added to above polymer base and subjected to stirring. Methyl paraben is added in small amounts as preservative and finally volume is made upto 10ml with distilled water¹⁵. (Note: Triethanolamine is added to form gel in q.s. and stirred well to obtain homogenous mixture)

EVALUATION PARAMETERS FOR NANOSPONGE LOADED GEL:

Determination of pH:

The pH of sertaconazole nanosponge loaded gel formulation was determined by using digital pH meter. One gram of gel was dispersed in 100ml of distilled water and stored for two hours at constant temperature. The measurement of pH was done in triplicate and average values were calculated.

Drug content:

Sertaconazole content in nanosponge gel was measured by dissolving 1000mg of gel in 10ml solvent (methanol) by sonication. The solution was passed through the whatmann filter paper no.42 and filtered. Absorbance was measured after suitable dilution at 261nm in UV - 1800 spectrophotometer.

Homogeneity:

It was determined by visual inspection for the appearance of gel and presence of any aggregates¹⁹.

Extrudability:

Pfizer hardness tester was used to study the extrudability. 20gm of gel was filled in aluminium tube. The plunger was adjusted in such a way to hold the tube properly. The pressure of 1kg/cm² was applied for 30 sec. The quantity of gel extruded was weighed. The procedure was repeated at three equidistance places of tube.

Spreadability:

By measuring the spreading diameter of 0.5g of gel between 20 x 20 cm glass plates after 1 min, spreadability of the formulated gel was determined. The mass of the upper plate was standardized at 500g¹⁶.

S = m * l * t

Where, S = Spreadability; m = weight applied to the upper glass slide; l = length of the glass slide; t = time taken in seconds.

Determination of viscosity:

Viscosity of prepared gels was determined by Visco Lab 3000 viscometer which contains a piston electromagnetic sensor and thermometer that provides viscosity and temperature readings respectively. Sample size of around 1 - 2ml was applied on the sensor in the chamber and the results were displayed on the screen of Visco Lab 3000. Determination was done in triplicates and the average was calculated.

Swelling studies:

Dried hydrogels were weighed accurately and kept immersed in 10ml of pH 7.4. Phosphate buffer. Hydrogels were taken carefully at different time intervals for 24 hours. Blotted with filter paper and weighed accurately. Increase in weight was determined as time increases. The percentage swelling was calculated from the equation¹⁷.

% Swelling = Wet weight – Dry weight / Wet weight * 100

In-vitro diffusion studies (dialysis membrane):

Diffusion studies were performed using Franz diffusion cell. The cell was locally fabricated and the volume of receptor compartment was 250ml. The dialysis membrane used for diffusion studies was placed between donor and receptor compartment. Sertaconazole loaded nanosponge gel was uniformly applied on membrane and clamped together. The receptor compartment was filled with pH 7.4 phosphate buffer and maintained by continuous stirring at 150rpm with a magnetic bead and maintained at 37˚C. At predetermined time intervals, 5ml samples were withdrawn and replaced with an equal volume of buffer. The samples were analyzed after appropriate dilution at λmax of 261nm using spectrophotometer. Release rate was calculated by plotting the amount of drug permeated Vs. square time. The slope is release rate (µg/cm²/hr½)¹⁹.

Ex-Vivo diffusion studies:

Ex-vivo diffusion studies were conducted for optimized formulation, marketed formulation (Onabet), pure drug dispersion and control gel using Franz diffusion cells at 350rpm for 8hrs. Male Wistar rats (150 - 180g) were used for permeation study. The animal was sacrificed by over anesthesia of isoflurane and hair was removed from abdomen using an animal hair clipper. Abdominal skin section was excised and observed for existence of cuts and wounds, washed under tap water. The skin was stored at −20°C and used within a week¹⁸. Permeation parameters such as percentage drug release, steady state transdermal flux (SSTF), permeability coefficient, lag time and skin retention of sertaconazole was calculated.

Skin deposition studies:

Skin deposition studies determined the amount of formulation retained in the skin. At the end of permeation studies (8th hr), skin was chopped into small pieces and left in methanol overnight. Measurable skin sample was homogenized with methanol for 15 - 30 min and the resulting solution was centrifuged at 6000rpm for 15 min and double filtered with Whatsmann filter paper. The supernatant was analyzed using UV-1800²⁰.

Calculation of model dependent kinetics for prepared nanosponge gel formulation:

Kinetics of drug release were tested using various models to analyze the mechanism of the drug release and rate kinetics of the dosage form. The obtained data was fitted into zero order, first order, Higuchi and Korsmeyer - peppas release model to study the drug release from the dosage form.

Skin irritation studies for the optimized nanosponge gel formulation:

Skin irritation study was performed by using control gel and test sample which were applied on right dorsal surface of rabbit skin and rabbits were examined for 72 hrs for erythema and edema. The score was given according to the Primary Dermal Irritation Index Classification (PDDI).

Stability studies for the optimized nanosponge gel formulation:

Stability studies were conducted for 1 month at room temperature, as the formulation contains all the excipients which are stable at room temperature. Furthermore, pure drug is not recommended to store at refrigerator conditions.

RESULTS AND DISCUSSION:

Analytical results of sertaconazole nitrate

Figure 2: FTIR of (A) pure drug and (B) optimized formulation

The IR spectrum is very specific to each chemical structure and provides structural information with references to peaks associated with characteristics group. The drug and optimized formulation characteristic peaks were shown in the above figures. The peaks observed in the FTIR spectrum of optimized formulation showed no shift and no disappearance of characteristic peaks suggesting no interaction between the drug and excipients i.e, the pure drug was not altered functionally and is compatible with excipients used in the formulation.

Conclusion from preliminary trails for the formation of nanosponges:

Among all the five polymers chosen, nanosponges were formed with ethyl cellulose and polymethyl methacrylate. So, these two polymers were selected for preparation of nanosponges and further studies were carried.

Optimization of drug and polymer ratios:

Among all the formulations T-5(76.33%) and T-11(84.87%) had good % EE than other formulations. So 1:5 ratio of drug and polymer was optimized for the preparation of nanosponges as this ratio entrapped more amount of drug with both the polymers. It was observed that as drug: polymer ratio increases particle size decreases and %EE increases. But, this is applicable only upto some extent, as drug: polymer ratio further increases %EE decreases as (1:6) ratio, this is due to polymer polymer interactions overruling than drug: polymer interactions¹³.

Optimization using Taguchi OA L⁸ design experiment

Eight formulations were obtained from Taguchi experimental bathes. Entrapment efficiency is conducted for all the eight formulations. The better entrapped formulations were selected for further studies. From the %EE studies F-2 (76.33%)), F-5 (84.55%) and F-8(84.87) were selected as these formulations have better %EE than others. Further, these three formulations were evaluated for % drug release for screening one formulation.

The P values of %EE Vs. type of polymers, concentration of surfactant and amount of dichloromethane is ˃ 0.5 which indicates it is statistical insignificance and accepts null hypothesis. This indicates that none of the ingredient effects the formulation and no difference will be observed due to sampling or any error

From the Main effect plot for means, it can be inferred that Factor B i.e., percentage of surfactant has greatest influence on the response. The other Factors A and C i.e., type of polymer, amount of dichloromethane has equal influence on the response. Therefore, the order of independent variables on which entrapment efficiency depends was percentage of surfactant > type of polymer > amount of dichloromethane.

In-vitro diffusion studies for optimization of formulations:

From the Taguchi experimental batches, three formulations (F2, F5 and F8) have shown good %EE and drug content, so these three formulations were tested in-vitro for their % drug release and were screened accordingly. The in-vitro drug release data is shown in figure-3. The formulations F2, F5 and F8 have shown 17.77%, 25.98% and 64.62% of drug release in 8 hours respectively. Formulation F2 have shown least amount of drug release as compared with other two formulations. From the above results, it was observed that formulation F2 is showing controlled action which contains ethyl cellulose, 0.2% of polyvinyl alcohol and 20ml of dichloromethane. So, F2 is screened from the Taguchi experimental batches.

Evaluation results of nanosponge formulation:

Surface morphology and the three - dimensional nature of the nanosponge studied through SEM, confirmed the presence of porous nature of sponges with smooth surface. Pores on the surface are formed due to evaporation of solvent (DCM) at high stirring speed. These pores act as channels for drug permeation into the skin through control manner.

Figure 3: Cumulative percentage drug release of optimized batches

Figure 4: SEM images of F2 formulation

Size distribution studies:

The vesicles obtained had a diameter of 169.5 ± 47.0 d.nm and the polydispersity index 0.312 indicating the screened formulation is polydisperse in nature. The particle size analysis revealed that the nanosponges were of nano size which is an ideal characteristic for the transdermal delivery of the drug. Nano sized particles have more surface area and increased skin retention achieving control release of drug.

Zeta potential:

Zeta potential was measured at Indian Institute of Chemical Technology, Hyderabad, India, using Malvern zeta sizer and the value found to be -12.4 mV. Negative charge on the particle may be due to the presence of methanol in the sample which prevents aggregation of sponges due to electrostatic repulsion ensuring stability of nanosponges and avoidance of particle aggregation.

Production yield:

Optimized formulation has percentage yield of 86% calculated by respective formula.

Entrapment efficiency:

Drug entrapment evaluates the potential of drug delivery into the system which is an important parameter. For this reason, the entrapment of sertaconazole within the formulation was evaluated to investigate the influence of nanosponge composition i.e., type of polymers, percentage of surfactant, and amount of crosslinking agent used on the drug loading capacity.

% Entrapment efficiencies of Taguchi experimental batches were performs, F-2 is screened as final formulation as it shows controlled effect when compared with F-5 and F-8. Furthermore, it has good %EE of 76.33%. Although the PMMA polymer have good %EE, it did not show good controlled effect compared with ethyl cellulose. Ethyl cellulose had good control release effect as it is insoluble rate retarding polymer. Percentage of surfactant plays a major role in particle size and %EE. As concentration of surfactant increases, less particle size nanosponges with low %EE are formed. It was also reported that with increase in surfactant concentration foaming is seen which leads to decreased filtration rate and increased synthesis time. By increasing crosslinking amount %EE is decreased due to increase in pore size.

Results of physicochemical evaluation of transdermal gels:

Table 4: Physicochemical evaluation of F-2 and control gel

Formulation	Optimized formulation (F-2)	Control gel
Drug content (%)	94.89±0.3	92.47±0.2
pH	6.57±0.6	6.88±0.8
Viscosity	6576	6367
Homogeneity	+++	++
Extrudability	+++	++
Spreadability (cm)	2.6±0.04	2.4±0.01
Swelling studies (%)	66.6%	75%

Note: All values expressed in mean ±SD, n=3

Results of ex-vivo diffusion studies:

The ex-vivo studies data for the optimized formulation (F-2), marketed formulation, control gel and pure drug release profile showed in Figure: 5. It was observed that formulation F2 showed the drug release of (11.27%), marketed formulation (63.58), control gel (98.21) and pure drug of (84.01%), which indicates F-2 formulation have low % drug release than other three formulations. So, it is an ideal formulation for delivering the drug in a controlled manner.

Figure 5: Cumulative percentage drug release of optimized formulation, marketed formulation, control gel and pure drug

Permeability parameters:

Table 5: Permeability parameters

Formulation code	Flux (µg/cm² /hr)	Permeability coefficient (cm/hr)	Lag time (hrs)
F-2	50.87	2.54	1.5
Marketed	270.55	13.5	0.5
Pure drug	432.33	21.61	0.1
Control gel	839.65	41.9	0.2

Skin retention studies:

The Ex-vivo skin deposition profile of sertaconazole nitrate from the optimized formulation (F-2) was found to be 70.40% whereas the skin deposition of marketed formulation, pure drug and control gel was found to be 49.23%, 11.47% and 6.37% respectively. Skin deposition showed high fold increase for the drug loaded nanosponges compared to other three formulations. Thus, the aim of the study is achieved which is formulating sertaconazole nitrate in a suitable formulation which decreases its permeability and increases percentage skin retention Nanosponges are known to be efficient vesicular system for delivering both hydrophilic and lipophilic drug.¹⁶

Model dependent kinetics:

From the results of ex-vivo drug release kinetics for all the above formulation, it was observed that optimized formulation (F-2) has zero order drug release kinetics, the drug release mechanism was found to follow Higuchi drug release mechanism, which indicates the rate of drug release through the mode of diffusion. From the value of release component “n” it can be concluded that the formulation is anomalous transport.

Skin Irritation studies:

The optimized formulation was stable at room temperature for 1 month without any physical change. Further no erythema or edema is seen after 72 hrs on rabbit skin, indicating nanosponge loaded gel (F-2) is non-irritant.

CONCLUSION:

Sertaconazole nitrate was successfully formulated into nanosponges loaded hydrogel for topical application. Drug and excipients were compatible with each other which was confirmed by FTIR.

Taguchi orthogonal array design (L⁸) was used to screen the formulation. Screened formulations were evaluated for percentage yield, %EE, particle size, zeta potential, drug content, spreadability, extrudability, viscosity, pH, homogeneity, in-vitro and ex-vivo studies and percentage skin retention studies.

In-vitro diffusion studies of screened formulations (F-2, F-5, F-8) revealed that F-2 has control release effect and optimized based on %EE, drug content and in-vitro diffusion studies. The average particle size, PDI, for screened formulation was found to be 169.5 nm, 0.312, respectively with zeta potential of -12.4mV which indicate that the formulation is nano in size and physically stable.

Ex-vivo permeation studies for screened formulation (F-2) have shown control release of drug with flux of 50.87µg/cm² /hr. Skin retention studies of screened formulation was found to be 70.40%, which indicate that more amount of the drug was retained in skin itself and less amount of the drug got permeated through the skin compared to marketed formulation (Onabet), pure drug and control gel.

Optimized formulation has no skin irritation which is stable at RT for 1 month. Statistical analysis of Taguchi results suggested that all the factors which are considered have no pronounced significant effect on response (Factors: type of polymer, percentage of surfactant and amount of dichloromethane). Hence, nanosponges act as a better carrier for sertaconazole nitrate to provide localized action to treat fungal infections while reducing its side effects and dosing frequency.

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Received on 22.10.2019 Modified on 29.01.2020

Research J. Pharm. and Tech. 2021; 14(2):895-902.

DOI: 10.5958/0974-360X.2021.00159.1

Material (% w/v)	F1	F2	F3	F4	F5	F6	F7	F8
Polymer	EC	EC	EC	EC	PMMA	PMMA	PMMA	PMMA
Percentage of Surfactant (%)	0.2	0.2	0.3	0.3	0.2	0.2	0.3	0.3
Amount of Dichloromethane(ml)	10	20	10	20	10	20	10	20
Solvent (ml)	150	150	150	150	150	150	150	150