FORMULATION DEVELOPMENT AND EVALUATION OF MUCOADHESIVE MICRO-SPHERES OF SALBUTAMOL SULPHATE BY USING A NATURAL POLYMER

FORMULATION DEVELOPMENT AND EVALUATION OF MUCOADHESIVE MICRO-SPHERES OF SALBUTAMOL SULPHATE BY USING A NATURAL POLYMER

Kavita N. Patil* and Kanchan P. Upadhye

J.L. Chaturvedi College of Pharmacy, Electronic Building, Hingna Road, Nagpur- 440 016, Maharashtra, India

ABSTRACT: Salbutamol  sulphate (SS)  loaded  microspheres  were  prepared   by  solvent  evaporation  method  with  combination   of  Sterculia  foetida  gum  in  various  proportions.  A total  of  eighteen  formulations  were  prepared  in  different  oil  phases  i.e.,  liquid  paraffin  and  sunflower  oil. Those  prepared  from  liquid  paraffin L1-L9 and  those  prepared  from  sunflower  oil  were  S1-S9.  Based on  the  particle size, entrapment  efficiency, drug  content,% yield, swelling  index, % moisture  loss, micromeritic properties batches  prepared  from  sunflower  oil showed better results as compared to those microspheres prepared from liquid paraffin. Thus, sunflower oil batches were optimized for further evaluation parameters. From further parameters four formulations were selected i.e., S2, S6, S8, S9. It  was  confirmed  with  the  results  of  micromeritic  property  that  all  the  selected  formulations   showed  good  flow  property.  Release  data  were  analyzed  based  on  best  fitting models  and  all  the  optimized batches  showed  good  fit  to  first  order,  Higuchi kinetics and  Korsemeyer-Peppas. Stability studies showed almost  negligible  changes  in  particle  size,  drug  content and  drug  release  throughout  the  study  period.

Salbutamol Sulphate, Natural Polymer, Controlled Release, Microspheres

INTRODUCTION:               Novel drug delivery systems [NDDS] that can precisely control the release rate or target drugs to a specific body site have had an enormous impact on the healthcare system. Microspheres constitute an important part of these particulate drug delivery systems by virtue of their small size and efficient carrier characteristics. However, the success of these novel drug delivery systems is limited due to their short residence time at the site of absorption.

It would, therefore, be advantageous to have means for providing an intimate contact of the novel drug delivery systems with absorbing membranes. Coupling mucoadhesion characteristics to microspheres and developing novel delivery systems as mucoadhesive microspheres can achieve it ¹.

Mucoadhesive drug delivery systems are one of the novel drug delivery system, which utilize the property of bioadhesion of polymers that become adhesive on hydration ². These drug delivery systems can be used for targeting a drug to a particular region of the body for extended period of time ³. Bioadhesion is an interfacial phenomenon in which two materials, at least one of which is biological, are held together by means of interfacial forces ⁴.

The attachment could be between an artificial material and biological substrate such as adhesion between a polymer and biological membrane. In case of polymer attached to the mucin layer of mucosal tissue, the term mucoadhesion is used. Mucoadhesive materials have been investigated and identified ⁵. These are generally hydrophilic macromolecules that contain numerous hydrogen bond forming groups (e.g. hydroxyl and carboxyl groups) and will hydrate and swell when placed in contact with water.

In most cases these materials require wetting to become adhesive. However, over hydration may result in the formation of slippery mucilage and a loss of adhesive properties. Mucoadhesive microspheres include microparticles and microcapsules (having a core of the drug) of 1-1000μm in diameter and consisting either entirely of a mucoadhesive polymer or having an outer coating of it, respectively ⁶.

Microspheres in general, have the potential to be used for targeted and controlled release drug delivery but coupling of mucoadhesive properties to microspheres as additional advantages e.g. efficient absorption and enhanced bioavailability of the drugs due to high surface to volume ratio, a much more intimate contact with the mucus layer. Mucoadhesive microspheres can be tailored to adhere any mucosal tissue including those found in eye, nasal cavity, urinary and gastrointestinal tract, thus offering the possibilities of localized as well as systemic controlled release of drugs. Various natural polymers have been used for preparing mucoadhesive microspheres.

Microspheres provide sustained release over a prolonged period of time and better bioavailability than conventional dosage forms which reduces dosing frequency, side effects and thereby increases patient compliance. The smaller size and spherical shape of microspheres increases the surface area which increases the bioavailability of the dosage form. It also has advantage over the microparticles and nano particles as they tend to accumulate at the site of action but microspheres due to its smaller size (i.e. micron size) and spherical shape can be injected and hence shows better bio-availability. Microspheres are defined as spherical microscopic particles having a size range of 1- 1000μm.

Salbutamol sulphate is a short acting beta–adrenergic agonist with more bronchodilatory effect and less cardiac stimulatory effect and useful in the treatment of bronchial asthma. Salbutamol sulphate is readily absorbed from gastrointestinal tract. The plasma half-life of Salbutamol sulphate varies from 2 to 7 hours. In the treatment of asthma, it is given in the dose of 2-4mg, three to four times a day orally.

MATERIALS AND METHODS: Salbutamol sulphate was obtained as a gift sample from Supriya Lifesciences Ltd., Mumbai. Sterculia foetida gum and sunflower oil was purchased from the local market. Liquid paraffin and Span 20 was obtained from Loba Chemicals, Mumbai. All other reagents used were of analytical grade.

Preparation of salbutamol sulphate microspheres using Sterculia foetida gum as a natural polymer: Microspheres were prepared by water in oil emulsification solvent evaporation technique. A polymeric aqueous solution was  made  adding drug  and  the  polymer  and  then  the  solution  poured  into  100 ml  of  light  liquid  paraffin containing 0.5% span-20 as an emulsifying  agent. The aqueous  phase  was  emulsified  in  oily  phase by stirring  the  system  in  a  500ml   beaker.  Constant stirring at 500-1000 rpm was carried out using mechanical stirrer. The beaker and its  content were heated at 250^oC using  heating  mantle, stirring  and  heating  were  maintained  for  2.5 hrs.  The aqueous phase was evaporated.  The  microspheres were washed with Iso-propyl  alcohol  or  n-hexane,  separated  and  dried  at  room  temperature.  The  same  procedure  was  done  by  changing  the  oil  phase  by  sunflower  oil.

Total 18 batches were prepared, 9 batches with  liquid  paraffin oil phase and 9 batches with  sunflower  oil  phase  with  different  drug: polymer  ratios . For all batches RPM and temperature were maintained constant. All the batches were selected for optimization and evaluated.

Constant stirring at 500-1000 rpm was carried out using mechanical stirrer. The beaker and its content were heated at 25^oC using heating mantle; stirring and heating were maintained for 2.5 hrs. The aqueous phase was evaporated.  The microspheres were washed with Iso-propyl alcohol or n-hexane, separated and dried at room temperature.

The same procedure was done by changing the oil phase by sunflower oil. Total 18 batches were prepared, 9 batches  with  liquid  paraffin  oil phase  and  9  batches  with  sunflower  oil  phase  with  different drug: polymer ratios. For all batches   RPM and temperature were maintained constant. All the batches were selected for optimization and evaluated.

TABLE 1: COMPOSITION OF SALBUTAMOL SULPHATE MICROSPHERES IN LIQUID PARAFFIN OIL PHASE

TABLE 2: COMPOSITION OF SALBUTAMOL SULPHATE MICROSPHERES IN SUNFLOWER OIL PHASE

FIG. 1: SCHEMATIC REPRESENTATION OF PREPARATION SALBUTAMOL SULPHATE MICROSPHERES BY SOLVENT EVAPORATION TECHNIQUE

Flow Properties: The flow properties of drug-loadedmicrospheres were investigated by measuring theangle of repose using fixed base cone method.The bulk and tapped densities were also measuredin a 10ml graduated measuring cylinder as ameasure of packability of the microspheres.

Theangle of repose (q) was determined by theformula, q = tan^-1(h/r); h=cone height ofmicrobeads; r = radius of   the circular base ¹⁰. Eachexperiment was carried out in triplicate.

Particle size analysis ⁷: The microsphere size   distribution was determined by the method using    a calibrated stage micrometer (μm). Different  sizes  in  a  batch  were  determined  by  MOTIC  PLUS – 2.0 ml  instrument. The average sizes of the microspheres were calculated.

Percentage yield: The dried microspheres of each batch are weighed separately and percentage yield is calculated by using following equation:

Percentage Yield=     Practical Weight    x 100

Theoretical Weight

Drug Content:50mg of mucoadhesive micro-spheres were weighed and powdered. This was dissolved in phosphate   buffer (6.8 pH) in 100 ml volumetric flask and made up to volume. The solution was shaken occasionally for 1h and filtered. From this, 1ml of solution was diluted upto 100 ml with pH 6.8 buffer solution in 100 ml volumetric flask. The drug content was  analyzed  by measuring absorbance in a UV spectrophoto-meter at  270  nm  using  pH 6.8 phosphate  buffer  as  blank.  The studies were carried out in triplicate. Drug content is calculated by the formula:

Drug Loading (%) = W_d x 100

W_m

Where, W_d is the weight of drug, W_m the weight of the microspheres.

Microencapsulation efficiency: 100mg of   muco-adhesive microspheres  were  accurately  weighed.  They were powdered and extracted with 100 ml of methanol.  Further, it was serially diluted with   pH 6.8 phosphate buffer solution. The resulting solution was analyzed for salbutamol sulphate drug content by measuring absorbance in a UV spectro-photometer at 270 nm using pH 6.8 phosphate buffer   as blank.  The   studies   were   carried out in triplicate. Encapsulation   efficiency (%) was calculated using the formula;

Encapsulation Efficiency=

Actual Drug Content        x 100

Theoretical Drug Content

Swelling Index: Swelling index  is  determined  by  measuring  the  extent  of  swelling  of  microspheres in a  phosphate  buffer   pH 6.8 . To ensure  the  complete  equilibrium,  exactly  weighed  100 mg of  microspheres  are  allowed to swell  in  buffer  for 34 hrs. The  excess  surface  adhered  liquid  drops are  removed  by  blotting  and the swollen  microspheres  are  weighed  by  using  microbalance. The  Hydrogel microspheres  then  dried  in  an  oven  at  60° for  5 hrs  until there is  no change  in  the  dried  mass  of  sample. The swelling index of the microspheres is  calculated  by  using  the  formula;

Swelling Index=

Mass of Swollen MS - Mass of Dry MS x 100

Mass of Dried MS

Percentage of moisture loss: The salbutamol sulphate loaded microspheres of different polymers were evaluated for percentage of moisture loss which sharing an idea about its hydrophilic   nature.  The microspheres weighed initially and kept in desiccators containing calcium chloride at 37°C for 24 hours. When no further change in weight of sample was observed, the final weight was noted down.

In-vitro mucoadhesion ⁸: The mucoadhesive  property  of  microspheres  was  evaluated  by  an in  vitro  adhesion  testing  method  known  as  wash  off  method.  Freshly excised  piece  of  intestinal  mucosa  (2 x 2 cm)  from  albino  rat  were mounted  onto  glass  slides  (3 x 1 inch)  with  cyanoacrylate  glue.

Two glass slides  were  connected  with  a suitable  support,  about  50  microspheres  were spread  on  to  each  wet  rinsed  tissue  specimen and immediately thereafter  the  support  was  hung  onto  the  arm  of  a  USP  tablet disintegrating  test  machine.

When the disintegrating test machine was operated,  the  tissue  specimen  was  given  slowly,  regular  up  and  down moment  in  the  test  fluid  (500 ml  pH 6.8  phosphate  buffer)  maintained  at 37º C.  At  the  end  of  30 min,  1h,  and  hourly  intervals  upto  6h,  the number  of  microspheres  adhering  to  tissue  were  counted.

In-vitro drug release studies ⁹: The release of Salbutamol sulphate from mucoadhesive micro-spheres was investigated in pH 6.8 phosphate   buffer   solution   as   a dissolution   medium   (900 ml)   using USP type I apparatus.  A  sample  of  microspheres  equivalent   to  50  mg  of salbutamol  sulphate   were  taken  in  the  muslin  cloth  tied  on  to  the  paddle of  apparatus.   A   speed of 50 rpm and temperature of 37±0.5^oC was maintained throughout the experiment. At fixed intervals,   aliquots (5 ml) was withdrawn and replaced with fresh dissolution   media.

The concentration   of   drug   released   at   different   time   intervals   was then determined by measuring the absorbance using Hitachi U-2000 spectrophotometer at 276 nm   against   blank. The studies were carried out   in   triplicate.  The   in vitro dissolution   data   of   oral   mucoadhesive microspheres   were tabulated   and calculated by using dissolution software viz., PCP DISSO V3.0.

In-vitro  drug  release kinetics: In order to study the exact mechanism of the drug release from microspheres, drug release data was analyzed according to Zero Order, First Order, Higuchi square root, Hixon Crowell, Korsemeyer model. The criterion for selecting the most appropriate model was chosen on the basis of goodness to fit test.

To analyze the mechanism for the release and release rate kinetics of the dosage form, the data obtained was fitted in to Zero order, First order, Higuchi matrix and Korsemeyer and Peppas model. Using PCP-DISSO – v2 software.  Comparing the r²-values obtained, the best-fit model was selected.

Scanning Electron Microscopy ¹⁰:The particle size, shape and surface morphology of micro-spheres were examined by scanning electron   microscopy (SEM). Microspheres were fixed on   aluminium   studs   and   coated   with   gold   using a sputter coater   SC 502, under Vacuum [0.1 mm Hg].  The microspheres   were then   analyzed   by   scanning   electron   microscopy (SEM) [Model JSM-840 A, Joel. Japan].

Drug – excipients compatibility studies:Drug-Excipients Compatibility were done by Fourier Transform Infrared Spectroscopy (FTIR).

Fourier Transform Infrared Spectroscopy (FTIR): The compatibility between pure drug and    polymers   were   detected   by IR   spectra   obtained   on   Perkin   Elmer 1600 series, (USA).   The   pellets were   prepared on    KBr–press.  To   prepare the pellets, a few mg of the   microspheres were ground together   in   a   mortar   with   about 100 times quantity of KBr. The   finely ground powder was introduced into a stainless   steel die.  The powder was then   pressed in the die between   polished   stainless   steel   anvils at a pressure   of   about   10t/in².   The   spectra   were   recorded over   the   wave   number   range   of 4000 to 500 cm-1.

Stability studies ¹¹: Selected microspheres   (formulation  code  SF 2, SF 6, SF 8, SF 9) were   wrapped  in  aluminium  foil  and  packed  in  glass  vials.  These  formulations  were  then  kept  in  an  incubator  maintained  at  40±0.50 C and  75±5% RH for  60  days. Changes  in  the  particle size , drug  content, drug  release and appearance of  these   stored  microspheres  were  investigated  at  regular   intervals  (0 days, 15 days , 30 days, 45  days, 60 days ).

RESULTS AND DISCUSSION:

Evaluation of Mucoadhesive Microspheres: All the evaluation parameters were performed with all the 18 batches. But from  the  results  of  micromeritic  properties, swelling  studies, drug  content, encapsulation  efficiency, % yield and  particle size of microsphere batches prepared in sunflower oil showed better than of the microsphere batches of liquid paraffin. Thus, the batches of microspheres prepared from sunflower oil were considered for further studies. Further from in vitro mucoadhesion and release studies four batches were optimized S2, S6, S8 and S9.

The Salbutamol sulphate (SS) microspheres were prepared by W/O Emulsion solvent evaporation technique by using natural polymer i.e. Sterculia foetida gum (SFG). Total 18 formulations were prepared. 9 formulations  were  prepared  from   sunflower  oil  and  9 formulations  were  prepared  from  liquid  paraffin  oil. The prepared formulations were then evaluated for different properties.

Micromeritic  properties: It  was  noticed  that  from  the  two  different  oil  batches, sunflower  oil  batches  showed  better  results  compared  to  liquid  paraffin  batches. The  microspheres  prepared  from  sunflower  oil  were  easily  washed compared  to  liquid  paraffin. They also showed excellent flowability this may be due to less viscosity of sunflower oil than liquid paraffin (table 3).

TABLE 3: MICROMERITIC PROPERTIES OF MICROSPHERES FROM SUNFLOWER OIL

 Particle  size  analysis :It  was  noticed  that the  particle  size  of  microspheres  increases  with  the  increased  concentration  of  Sterculia  foetida  and  this  may  be  due  to  high  viscosity  of  Sterculia  foetida  gum which  increases  the  droplet  size and  results  in  increase  particle  size (fig. 2, table 4).

Percentage  yield:  It was noticed  that  %  yield  of  microspheres  prepared  from  sunflower  oil  has  the  maximum  yield  compared  to  batches  prepared  from  liquid  paraffin (table 5).

Drug  content: The  drug  content in  microspheres using  sunflower  oil  as oil  phase  was  more  than  that  with  liquid  paraffin batches S7  showed  highest  drug  content (table 5).

Microencapsulation efficiency: Micro-encapsulation efficiency was more with sunflower oil as oil phase showed good microencapsulation efficiency with maximum microencapsulation efficiency of 96.8% with batch S9. The entrapment efficiency is increased with the lower concentration of  Sterculia  foetida  polymer (table 5).

Swelling  property : The  swelling  indices  increase  with  the  increase  in  concentration  of  Sterculia  foetida gum.  It  was  noticed  that  the  swelling  indices of  the  microspheres   prepared  from  sunflower  oil  were  high  as  compared  to  microspheres prepared from the liquid  paraffin (fig. 3).

% Moisture loss: The % moisture loss was determined for all the formulations prepared   from sunflower oil and liquid paraffin. It was noticed that the microspheres prepared from sunflower oil show minimum Percent moisture loss as compared to Microspheres prepared from liquid paraffin (fig. 4)

In-vitro mucoadhesion test: The no. of  micro-spheres   adhering  to  the  tissue  were  calculated  after  2hr, 4hr, 6hr, 8hr. After  determination  it  was found that  batch S9 showed  highest  percent  78%  mucoadhesion  than  other  batches. Three batches showed comparatively higher muco-adhesion in the following order S6<S8<S9 (fig. 5).

In-vitro  release  studies:  The  maximum  in-vitro  drug  release  was  found  to  be  99.77%  for  formulation  S9  at 12th hour. Formulation   S6, S8 showed maximum release of 95.8%, 97.07%   respectively at 12th hour. The release data were analyzed on the basis of best fitting models.  The release rates k and n of each model were calculated by linear regression analysis. Coefficients of correlation (r²) were used to evaluate the accuracy of the fit (table 6, fig. 6).

FTIR Studies: FTIR Spectral analysis was performed to study the drug polymer interaction.It was concluded that there were no changes in the peak shape and no shift of peaks. Thus, there  is  no  interaction  between  drug  polymer.  So they are compatible with each other (fig. 7-12).

Scanning electron microscopy: SEM of the selected formulation was performed. All  the  selected  microspheres  loaded  with  salbutamol  sulphate  were  smooth, almost  spherical, and  uniform.(fig 13-16)

Accelerated stability studies: Accelerated stability study data of the medicated microspheres are shown in table 7. At the initial  level and at the final level  of  the  accelerated  stability  study, the  tested  microspheres  showed  almost  similar  particle  size, drug  content and  drug  release  were  observed. No color changes or unexpected change were observed. All the optimized  batches were  found stable after 2 months without much  variation  in  particle  size,  drug  content, drug  release.

Micromeritic properties of prepared Mucoadhesive Microspheres:

Physical Characterization of Prepared Microspheres

TABLE 4:  PARTICLE SIZE AND SHAPE OF MICROSPHERES PREPARED FROM SUNFLOWER OIL

FIG. 2: PARTICLE SIZE ANALYSIS OF SALBUTAMOL SULPHATE WITH STERCULIA FOETIDA GUM USING LIQUID PARAFFIN AND SUNFLOWER OIL BATCHES

Percentage yield, Drug Content and Percent Drug Entrapment:

TABLE 5: PERCENTAGE YIELD, DRUG CONTENT AND PERCENT DRUG ENTRAPMENT OF MICROSPHERES FROM SUNFLOWER OIL (Mean ± S.D., n=3)

 Percent Moisture Loss and Swelling Index:

FIG. 3: % SWELLING OF SUNFLOWER OIL BATCHES

FIG. 4: % MOISTURE LOSS OF SUNFLOWER OIL BATCHES   

In-vitro Mucoadhesion test:

FIG. 5: MUCOADHESION BEHAVIOR OF MUCOADHESIVE MICROSPHERES FORMULATIONS PREPARED IN SUNFLOWER OIL pH 6.8

FIG. 6: IN-VITRO DRUG RELEASE OF SALBUTAMOL SULPHATE FROM S2, S6, S8, S9 BATCHES

 In-vitro drug release studies:

 TABLE 6: IN VITRO DRUG RELEASE DATA OF SALBUTAMOL SULPHATE MICROSPHERES WITH STERCULIA FOETIDA (Mean ± S.D., n=3)

 Drug-polymer interaction studies:

FTIR Studies: The FTIR spectrum of Salbutamol sulphate and Sterculia foetida gum alone and the Optimized batches of Microspheres were presented in figures:

FIG. 7: INFRARED SPECTRUM OF SALBUTAMOL SULPHATE

FIG. 8: INFRARED SPECTRUM OF STERCULIA FOETIDA GUM

FIG. 9: INFRARED SPECTRUM OF S6 BATCH

FIG. 10: INFRARED SPECTRUM OF S8 BATCH

FIG. 11: INFRARED SPECTRUM OF S9 BATCH

FIG. 12: INFRARED SPECTRUM OF S2 BATCH

Scanning Electron Microscopy:

FIG. 13: S2 BATCH

FIG. 14: S6 BATCH

FIG. 15: S8 BATCH

FIG. 16: S9 BATCH

Accelerated Stability Studies:

TABLE  7: PHYSICAL  STABILITY  CHARACTERISTICS  OF  SELECTED  SALBUTAMOL  SULPHATE  MICROSPHERES: (Mean ± S.D; n = 3)   Condition :  room  temp: 40^o C , relative  humidity:75%

 CONCLUSION: From the above experimental results, it can be concluded that:

• Oral controlled release of Salbutamol sulphate can be achieved by solvent evaporation technique using Sterculia foetida as a polymer.
• The  IR  spectra’s  revealed  that,  there  was  no  interaction  between  polymer and  drug. The entire polymer used was compatible with the drug.
• Prepared microspheres exhibited   Korsemeyer-Peppas kinetics/Higuchi model and  the  release  profile  was  by  Fickian and Anomalous
• From the study, it is evident that a promising controlled release microparticulate drug delivery of salbutamol sulphate can be developed. Further, in-vivo investigation is required to establish efficacy of these formulations.
• The study  also indicated  that  the  amount of  drug  release decreases with an increase  in  the  polymer  concentration .
• Finally, it can be concluded that the  micro-spheres of salbutamol sulphate can offer the  following  potential  advantages:

1. Reduced  frequency  of  administration
2. Increased  therapeutic  response
3. Improve  patient  compliance
4. Improved flow and packing property.

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How to cite this article:

Patil KN and Upadhye KP: Formulation development and evaluation of Mucoadhesive Microspheres of Salbutamol sulphate by using a Natural polymer. Int J Pharm Sci Res 2013; 4(12): 4775-86. doi: 10.13040/IJPSR. 0975-8232.4(12).4775-86

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