INTRODUCTION:

In pharmaceutical inhalation aerosols, recent advances in nanotechnology particularly in the area of drug delivery for systemic drugs, is technically challenging as variations in inhalation techniques can affect quantity of drug delivered to the lungs. The aerosol technology has clearly demonstrated its effectiveness for therapeutic application in treatment of pulmonary diseases. Various studies ^1,2 have illustrated that liposomal aerosols as nanoenabled aerosols are more effective for delivery, deposition and retention of drugs in pulmonary region. As inhalation aerosols provide a high drug concentration in broncho alveolar fluids and other lung tissues when presented as oral inhalation products³. For this reason pulmonary delivery is the most useful drug presentation for lung diseases. Asthma and chronic obstructive pulmonary diseases are common diseases of the airways and lungs that have a major impact on health of population. The mainstay of treatment is by inhalation of medication to the site of disease process ⁴.

Nebulisers are useful for giving high doses of medication but are generally prescribed only for people with severe asthma. Nebuliser pumps pressurise air through liquid medication to convert it into fine vapour which is then breathed through a mask or a mouthpiece. Medicines such as preventers, relievers, antibiotics such as tobramycin, gentamicin and carbenicillin are found to be successful in the treatment of cystic fibrosis of lungs.⁵ Nebulisers are also used for clearing sputum. Ultrasonic nebulisers (aerosonic nebulisers) nebulise medicines very fast using ultrasonic sound waves in the conversion of liquid medicines. Modern ultrasonic nebulisers use innovative mesh technology with vibrating membrane to produce fine aerosol for fast and effective aerosol therapy. Drug delivery rate and deposition pattern in the lungs play an extremely important role in efficacy of drug as deposition in upper airways, oesophagus and mouth shows unreachable fraction. The flow of compressed air should be 6-8litres per minute to drive the nebuliser. Drug inhalation is accomplished by normal tidal breathing over 10-15 minutes period by deep and usually rapid inhalation.

Liposome encapsulated drug delivery to lower respiratory tract through aerosol delivery has been proved to be effective in treating various pulmonary conditions. Drugs used in chronic obstructive pulmonary disorders such as salbutamol, terbutaline or ipratropium (Atrovent) at a higher dose can be used in liposomal dispersions in nebulising solutions. High drug loading and stability is to be considered in liposomal formulation. Liposomes must retain the drug in the lung for a sufficient period of time to be available for targeted release and minimise dosing requirements. Because of leaky pulmpnary membranes and large blood flow through lungs drugs in solution are generally absorbed in a relatively short period of time. Drugs with low lipophilicity dissolve in the lung very quickly and consequently are rapidly absorbed into systemic circulation, as liposomal membrane is comprised of saturated lipid phosphatidyl choline and cholesterol which are the natural constituents of lung surfactant. These lipids are not only biocompatible but also impart a high degree of membrane stability due to factors including suppression of gel to liquid crystalline phase transition thus making nebulised delivery feasible and maintaining a high localised concentration of drug in the lung. Nebulisers are aqueous preparations and cosolvents such as glycerine, propylene glycol and ethanol may be used. Nebulising solution needs to be iso-osmotic as hypertonic or hypotonic solutions cause chest tightness and bronchoconstriction giving poor compliance⁶. Osmolarity increases during nebulisation from 11 to 62% hence it is suggested to dilute the drugs with 0.9% saline and to restrict nebulisation time to ten minutes⁷.

MATERIALS AND METHODS:

Phospholipon^® 90G was procured from Natterman Phospholipid GmBH, and chloroform AR from Qualigens fine chemicals, salbutamol sulphate was a Gift sample from Midas Care Pvt. Limited, Distilled water was as per I.P, Cholesterol pure grade was obtained from BDH, α- tocoferol was from Sigma laboratories.All other chemicals and solvents used during experimentation were of analytical grade. Marketed preparation Asthalin was purchased from a local market.

Method of Preparation:

The liposomal dispersion was prepared as follows –

S. No.	Ingredients	Quantity
1	Phospholipon G	1.00g
2	Cholesterol USP	0.25g
3	α-Tocoferol USP	0.01g
4	Chloroform	100ml
5	Salbutamol sulphate I.P	0.100g
6	Phosphate Buffer pH 6.4 I.P q.s	100ml

Procedure:

Liposomes of salbutamol sulphate were prepared by Thin Film Hydration method as per the above formula. The lipid, Phospholipon G, cholesterol and α-Tocoferol were dissolved in chloroform in a round bottom flask (RBF). The flask was mounted on a rotary evaporator maintained at 40^oC and was rotated at a slow speed under vacuum to dry the film completely.The flask was flushed with nitrogen and Phosphate buffer pH 6.4 and the RBF was rotated for 1 hour for a complete hydration of the film in a water bath, maintained at 50^oC. Volume was made to 100ml with the buffer. The solution was filled in glass vials.

The optimised formulation was confirmed for isotonicity. Isotonicity was adjusted with the help of sodium chloride. Sodium chloride equivalent was calculated for individual component. and then extra sodium chloride required to adjust tonicity was determined. Sodium chloride was added as a component of phosphate buffer.

Evaluation of the Liposomal Dispersion:

Three identical batches of salbutamol sulphate liposomes of nebulizing solution were prepared (B. No. 01, 02, 03) .

The liposomal dispersion was studied for its physical appearance, texture, settling rate and redispersion time (Table- 1). The dispersion was observed for physical properties for a period of three weeks. Optical microscopy was carried out to confirm the bilamellar structure of liposomes and their size. (Table- 1). The particle size distribution was determined by measuring the size of the vesicles in 25 different fields, with the help of stage micrometer. pH was checked( Table -1). Size of the liposomal vesicles was determined by Laser diffraction method using Malvern Mastersizer. (Table-2 ) Fig - 1 shows the picture of the salbutamol liposomes under electron microscope.

Fig. 1 Electron Micrograph of Liposomal Nebulizing Dispersion of Salbutamol Sulphate

TABLE – 1: Study of Physical Characteristics of Salbutamol Liposomal Nebulizing Dispersions.

S. No.	Test	B. No. 01..1 of sodium chloride a	B. No. 02	B. No. 03
1	Physical Appearance	White colour, fine, uniform suspension	White colour, fine, uniform suspension	White colour, fine, uniform suspension
2	pH	6.42	6.38	6.44
3	Optical Microscopy	1-3µm with 2-3 bilayers.	1-3µm with 2-3 bilayers	1-3µm with 2-3 bilayers

TABLE – 2: Particle Size Analysis and Size Distribution Study of Salbutamol Liposomes by Laser Diffraction Technique.

B. No.	Sample No.	D10% (µm)	D50% (µm)	D90% (µm)
01	1	0.65	1.32	2.62
	2	0.84	1.36	2.21
	3	0.78	1.82	2.48
02	1	0.77	1.31	2.11
	2	0.68	1.56	2.36
	3	0.81	1.89	2.75
03	1	0.28	1.69	2.47
	2	0.62	1.34	2.70
	3	0.56	1.88	2.66
Average		0.67	1.57	2.48
Std. Dev. (±)		0.172	0.251	0.223

The amount of salbutamol sulphate entrapped (Percent Drug Entrapped) within the liposomes was estimated by spectrophotometry at 464nm. The results were studied for six samples of each batch. (Table-3 ). To determine Total Drug Content to 5ml of the liposomal nebulising solution, 1ml of ethyl alcohol was added and the solution was mixed on a vortex mixer. The content of salbutamol sulphate was estimated by spectrophotometry at 464nm. (Table-4 ). The in vitro diffusion studies were performed to check the rate at which drug is released from liposomes by determining diffusion of the drug entrapped in liposomes through a dialysis bag. (Graph-2).

TABLE – 3: Percent Drug Entrapment of Salbutamol Sulphate in Liposomal Nebulising Dispersions.

Sample No.	Percent Drug Entrapped
Sample No.	B. No. 01	B. No. 02	B. No. 03
1	48.56	51.09	49.10
2	47.92	50.88	47.72
3	47.61	48.63	47.24
4	48.39	49.1	50.68
5	50.14	50.26	48.90
6	48.11	48.17	48.66
Average	48.46	49.69	48.72
Std. Dev. (±)	0.891	1.223	1.201

TABLE – 4: Total Salbutamol Sulphate Content in Liposomal Nebulising Dispersions.

Sample No.	Total Salbutamol Sulphate Content (%)
Sample No.	B. No. 01	B. No. 02	B. No. 03
1	99.62	100.18	100.27
2	100.04	100.38	99.40
3	100.22	99.24	98.91
4	99.86	100.30	100.52
5	101.10	99.12	100.60
6	100.56	99.84	101.11
Average	100.23	99.84	100.14
Std. Dev. (±)	0.531	0.547	0.821

IN-VITRO DEPOSITION STUDIES:

Prerequisite for therapeutic efficacy is the ability of the aerosolised drug to reach the peripheral airways. The fundamental problem of inhalation therapy is that the anatomy and physiology of the respiratory tract is designed to prevent entry of particulate matter ^8,9.

The extent of aerosol deposition in the lungs is difficult to predict because particles can come in vastly different shapes and sizes. To account for shape and size, aerosol particles are often described by their aerodynamic diameter ¹⁰ . This diameter is related to particle size and density and is equivalent to the diameter of a perfect sphere of unit density (1g /cm³⁾ with the same settling velocity in still air ¹¹.

The deposition of inhaled aerosols is governed by the physical principles that apply to particles of different sizes. Three main processes of aerosol particle behavior in respiratory tract are inertial impaction, gravitational sedimentation and Brownian diffusion ¹².

Generally particles greater than 5μm are unable to follow the abrupt changes in the direction of the air stream during inspiration and impact in the upper airways of the respiratory tract. Smaller particles in the 1-5μm range settle in the lower airways and may reach the alveoli by gravitational sedimentation. Submicron particles behave like a gas and can deposit in the alveoli but are also readily exhaled ^13,14.

Although particle size is the most important determinant in aerosol deposition, the efficiency of deposition is influenced by a number of given factors –

i. Solutions containing salts or chemical substances that exert an osmotic effect can affect the stability and behavior of droplets once aerosolized. Hypertonic particles can attract water molecules, grow in size and deposit more proximally while particles of low tonicity may evaporate and shrink. Droplets can also collide and coalesce ¹⁵ .

ii. Compromised lung function in disease states such as obstructive lung disease ¹⁶ and asthma can restrict the dose of aerosol reaching the peripheral airways.

Since pharmacological effect of a drug depends on the dose fraction reaching the various locations in the respiratory tract, it is divided into the oropharyngeal region, tracheobronchial region and pulmonary region. The fraction of the dose depositing in pulmonary region, is considered as respirable fraction of aerodynamic diameter.

The in-vitro deposition pattern of the salbutamol sulphate liposomal nebulizing solution was determined with the help of Twin Impinger Apparatus, manufactured by ‘Copley’ and supplied by ERWEKA, Germany.

This simple two stage impinger with defined throat geometry is used for evaluating the total percentage of the active ingredient depositing in the two stages. The unit operates on the principle of liquid impingement to divide the dose emitted from inhaler into the non-respirable dose impacting on the mouth and oropharynx, which is swallowed and the remaining respirable dose. The two stages of the apparatus are designed such that airflow at the rate of 60 lit/minute through the system is obtained. The effective [mean] aerodynamic particle cut-off size of the lower impinger is 6.4μm.

Both the stages use the vertical impingement of the air stream on the liquid surface to form a convenient ‘trap’ for drug particle. The aerosol cloud deposits on the main impaction surfaces:

i. The back of the glass throat and upper impingement chamber: Stage I: Non-respirable dose.

ii. The lower impingement chamber which collects the remaining drug particle: Stage II: Respirable dose.

Method:

The two collecting chambers (stage I and stage II) were filled with 7ml and 30 ml respectively of the analyzing liquid (distilled water). The nebulizing device ‘CITIZEN Ultrasonic Nebulizer was attached by means of rubber collar. The nebulizing dispersion of liposomal salbutamol sulphate was nebulised for three minutes into the apparatus.

The ultrasonic nebuliser used was of CITIZEN make produces spray by piezoelectric crystal vibrating at high frequency. It consists of a cup for loading the liquid to be nebulised. The bath was filled with water and the cup is placed in the slot meant for the liquid to be nebulised. The liquid can be nebulised at three delivery rates. The side arm tube was connected to the vacuum pump, which was operated at a flow rate of 60 lits/min., which mimics the respiratory and inspiratory flow rate exerted by a normal individual. These reservoirs were rinsed with the analyzing liquid and the drug deposited on the collar, device, stage-I and stage-II was estimated by the spectrophotometric analysis.

The deposition of the drug at the device collar and the stage-I represents the fraction that is not available for inhalation ( non-respirable fraction).

The in-vitro studies were carried out with the three batches of nebulizing liposomal dispersions of salbutamol sulphate along with the two batches of marketed nebulizing solution, ‘Asthalin’.

The results of the in-vitro deposition pattern obtained from a liposomal nebulizing solution of salbutamol were compared with conventional salbutamol sulphate nebulizing solution. ( Graph 2).

Analysis of salbutamol sulphate from the nebulizing solution with Twin Impinger

3ml of the liposomal salbutamol sulphate nebulizing solution was put in the delivery cup of the nebulizer. The nebulizer was run at ‘high’ speed for 5 minutes along with the set up discussed earlier. The device, collar, stage-I and stage-II were rinsed with distilled water. The rinsing was collected and made to 25ml with distilled water. The absorbance was checked at 276nm.

A standard solution was prepared by weighing 100 mg. of salbutamol sulphate. This was dissolved in 100ml. distilled water (solution ‘a’). An aliquot of 10 ml of solution ‘a’ was diluted to 50 ml (solution ‘b’) with distilled water. 10 ml of the solution ‘b’ was further diluted to 25 ml with distilled water solution ‘c’. Absorbance of solution ‘c’ was checked at 276 nm against distilled water as a blank.and % salbutamol sulphate was determined.

The percent salbutamol sulphate deposited in device stage I and stage II of Twin Impinger was estimated in three samples for the three batches of the liposomal nebulizing solution of salbutamol sulphate.(Table-4)

RESULTS AND DISCUSSION:

All the three batches 01, 02 and 03 appeared as white coloured dispersion with very slow settling and excellent redispersibility and the pH of all the three batches was in the range 6.3 - 6.5 ( Table -1.)

The liposomal dispersions showed particle size in the range of 1-3µm with 90% of the particles below 3µm. (Table- 2 and Graph - 1.) Round vesicles with 2-3 striations on the periphery of the vesicles, confirming the lamellar structure of the liposomes were observed under the microscope. The vesicle size, for all the three batches 01, 02 and 03 in the range of 1 - 3µm. (Table- 1).The electron micrographs (Fig-1) showed bilamellar structure of the liposomes, in size range 1-3µm. The spectrophotometric analysis for percent drug entrapment showed entrapment of salbutamol sulphate in the range of 48-52%, (Table-3) this confirmed the uniformity and batch to batch reproducibility of dispersion.

As in the formulation, entrapped drug was not separated from the unentrapped drug because the unentrapped drug can produce the instant therapeutic effect, the total drug content was estimated. Each formulation contained 10mg of salbutamol sulphate per ml of the nebulizing dispersion. Salbutamol sulphate content was in the range 99-102%, for all the three batches 01, 02 and 03 (Table- 4).

The in vitro diffusion study shows that the unentrapped portion was available for its immediate effect within first two hours. 65.70% of salbutamol sulphate was released at the end of 6 hrs, 85- 90% by the end of 8 hrs and by the end of 9 to 10 hrs all the drug was completely released.( Graph- 2).

TABLE – 5: Net Respirable Fraction of Liposomal Nebulizing Dispersions of Salbutamol

Batch No.	Sample No.		% Salbutamol Sulphate deposited
Batch No.	Sample No.		Device	Stage I	Stage II	Mass balance
01	1		73.705	3.9578	23.626	101.29
	2		74.848	2.791	22.489	100.129
	3		72.719	3.054	23.850	99.623
02	1		74.123	2.012	25.755	101.89
	2		76.432	2.886	22.376	101.694
	3		76.018	3.116	23.159	102.293
03	1		73.105	1.996	24.206	99.307
	2		76.898	2.315	22.312	101.525
	3		77.661	2.985	21.679	102.32
Average			75.057	2.790	23.272	101.119
Std. Dev. (±)			1.67	0.62	1.24	1.14
Marketed Sample (Asthalin) Respirator Solution B.No.G20277		1	75.921	5.876	18.563	101.36
		2	76.622	6.560	18.854	102.036
		3	74.877	6.792	19.112	100.781
Average			75.81	6.41	18.84	101.39
Std. Dev. (±)			0878	0476	0.275	0.628

The in-vitro deposition studies indicate salbutamol sulphate nebulizing solutions by Twin Impinger method. The liposomal dispersions showed maximum i.e. 72-77% of deposition in the device, about 2-4% of salbutamol sulphate was deposited in the stage I, (upper respiratory tract deposition). Stage II showed 20-24% drug deposition, representing the available Net Respirable Fraction. (Table-5 ) The marketed conventional nebulising solution showed comparatively less Net Respirable Fraction, avg. 18.8% and more deposition in the upper respiratory tract, average 6.4%, as compared to the formulated liposomal preparations.

CONCLUSION:

Nebulizing solution of salbutamol sulphate was prepared in phosphate buffer 6.4. The solution was very slightly hypertonic or almost isotonic. The percent isotonicity was 1.08 and no extra sodium chloride was required for the further adjustment of the isotonicity.All the preparations were white coloured dispersions with excellent redispersibility and slow settling rate. The pH was in the range 6.3-6.5. Optical microscopy confirmed bi-tri lamellar structure with vesicle size in the range 1-3 µm. The results were confirmed by laser light scattering method. 90% of the vesicles were found below 3µm.

48-50% of salbutamol sulphate was found to be entrapped within the liposomes. Total drug content was 99-102%. The release was extended for almost 9 hrs from the liposomes.

The liposomal nebulizing solution of salbutamol sulphate showed average net respirable fraction 23.27% which was comparable with the marketed plain nebulizing solution preparation showing 18.84% average net respirable fraction. In both the cases the deposition in the device was very high i.e.75%. The liposomal preparation showed improved Net Respirable Fraction as compared to the marketed conventional nebulizing solution. The marketed preparation ‘Asthalin Respirator Solution’ had 18-19% of the Net Respirable Fraction and the deposition in the upper respiratory tract was about 6%.

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Received on 09.06.2011 Modified on 16.06.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1373-1378