1. FORMULATION AND EVALUATION OF MICROSPHERES
INTRODUCTION:
• A well designed controlled drug delivery system can overcome some of the problems
of conventional therapy and enhance the therapeutic efficacy of a given drug.
• To achieve maximum therapeutic efficacy, it becomes necessary to deliver the agent
to the target tissue in the optimal amount in the right period of time there by causing
little toxicity and minimal side effects.
• There are various approaches in delivering a therapeutic substance to the target site in
a sustained controlled release fashion. One such approach is using microspheres as
carriers for drugs.
• Microspheres are characteristically free flowing powders consisting of proteins or
synthetic polymers which are biodegradable in nature.
• Microspheres are defined as “Monolithic sphere or therapeutic agent distributed
throughout the matrix either as a molecular dispersion of particles” or can be defined
as structure made up of continuous phase of one or more miscible polymers in which
drug particles are dispersed at the molecular level or macroscopic level.
• It has particle size of 1-1000 nm.
• Due to smaller particle size it spreads to a large area in gastrointestinal tract which
improves drug absorption and reduces side effects due to localized buildup of
irritating drugs against the gastrointestinal mucosa.
TYPES OF MICROSPHERES:
• Bio-adhesive microspheres:
Adhesion can be defined as sticking of drug to the membrane by using the sticking
property of the water soluble polymers. Adhesion of drug delivery device to the
mucosal membrane such as buccal, ocular, rectal, nasal etc can be termed as bio-adhesion.
These kinds of microspheres exhibit a prolonged residence time at the site
of application and causes intimate contact with the absorption site and produces better
therapeutic action.
• Magnetic microspheres:
This kind of delivery system is very much important which localises the drug to the
disease site. In this larger amount of freely circulating drug can be replaced by smaller
amount of magnetically targeted drug. Magnetic carriers receive magnetic responses
to a magnetic field from incorporated materials that are used for magnetic
microspheres.
• Floating microspheres:
In floating types the bulk density is less than the gastric fluid and so remains buoyant
in stomach without affecting gastric emptying rate. The drug is released slowly at the
2. desired rate and increases gastric residence and fluctuation in plasma concentration. It
also reduces chances of sticking and dose dumping.
• Radioactive microspheres:
In Radio emobilisation therapy microspheres sized of 10-30 nm are larger than
capillaries and gets tapped in first capillary bed when they come across. They are
injected to the arteries that lead to tumour of interest. In all these conditions
radioactive microspheres deliver high radiation dose to the targeted areas without
damaging the normal surrounding tissues.
• Polymeric microspheres:
The different types of polymeric microspheres can be classified as follows:
a) Biodegradable polymeric microspheres:
Natural polymers such as starch are used with the concept that they are biodegradable,
biocompatible, and also bio adhesive in nature. Biodegradable polymers prolongs the
residence time when contact with mucous membrane due to its high degree of
swelling property with aqueous medium, results gel formation.
b) Synthetic polymeric microspheres:
The interest of synthetic polymeric microspheres are widely used in clinical
application, moreover that also used as bulking agent, fillers, embolic particles, drug
delivery vehicles etc and proved to be safe and biocompatible. But the main
disadvantage of these kinds of microspheres, are tend to migrate away from injection
site and lead to potential risk, embolism and further organ damage.
METHODS OF PREPARATIONS
Different methods used for various microspheres preparation depends on particle size,
route of administration, duration of drug release and these above characters related to
rpm, method of cross linking, drug of cross linking, evaporation time, co-precipitation
etc. The various methods of preparations are
1. Emulsion solvent evaporation technique
In this technique the drug is dissolved in polymer which was previously dissolved
in chloroform and the resulting solution is added to aqueous phase containing
0.2% sodium of PVP as Emulsifying agent. The above mixture was agitated at
500 rpm then the drug and polymer was transformed into fine droplet which
solidified into rigid microspheres by solvent evaporation and then collected by
filtration and washed with demineralised water and desiccated at room
temperature for 24 hours. Diclofenac microspheres are prepared by this method.
2. Emulsion cross linking method
In this method drug was dissolved in aqueous gelatine solution which was
previously heated for 1 hour at 40oC. The solution was added drop wise to liquid
3. paraffin while stirring the mixture at 1500 rpm for 10 min at 35oC, results in w/o
emulsion then further stirring is done for 10 min at 15oC. Thus the produced
microspheres were washed respectively three times with acetone and isopropyl
alcohol which then air dried and dispersed in 5 mL of aqueous glutaraldehyde
saturated toluene solution at room temperature for 3 hours for cross linking and
then was treated with 100 mL of 10 mm glycerine solution containing 0.1% w/v
of tween 80 at 37oC for 10 min to block unreacted glutaraldehyde. Examples for
this technique is Gelatin A microspheres.
3. Co-acervation method
a) Co-acervation thermal change:
Performed by weighed amount of ethyl cellulose was dissolved in cyclohexane
with vigorous stirring on the above solution and phase separation was done by
reducing temperature and using ice bath. Then above product is washed with
cyclohexane and air dried then passed through sieve (sieve no. 40) to obtain
individual microcapsule.
b) Co-acervation non solvent addition:
Developed by weighed amount of ethyl cellulose was dissolved in toluene
containing propyl isobutylene in closed beaker with magnetic stirring for 6 hours
at 500 rpm and the drug is dispersed in it and stirring is continued for 15 mins.
Then phase separation is done by petroleum benzoin with continuous stirring.
After that the microcapsules were washed with n-hexane and air dried for 2 hours
and then in oven at 50oC for 4 hours.
4. Spray drying technique
It involves dispersing the core material into liquefied coating material and then
spraying the mixture in the environment for solidification of coating followed by
rapid evaporation of solvent. Organic solution of poly (epsilon-caprolactone)
(PCL) and cellulose acetate butyrate (CAB), in different weight ratios and
ketoprofen were prepared and sprayed in different experimental condition
achieving drug loaded microspheres.
5. Emulsion-solvent diffusion technique
The drug polymer mixture was dissolved in a mixture of ethanol and
dichloromethane (1:1) and then the mixture was added drop wise to sodium lauryl
sulphate (SLS) solution. The solution was stirred with propeller type agitator at
room temperature at 150 rpm for 1hour. Thus the formed floating microspheres
were washed and dried in a desiccator at room temperature. The following micro
particles were sieved and collected.
6. Multiple emulsion method
In the beginning powder drug was dispersed in solution (methyl cellulose)
followed by emulsification in ethyl cellulose solution in ethyl acetate. The primary
4. emulsion was then re emulsified in aqueous medium. Under optimised condition
discrete microspheres were formed during this phase.
7. Ionic gelation
Alginate/chitosan particulate system for Diclofenac release was prepared using
this technique. 25% (w/v) of Diclofenac was added to 1.2 % (w/v) aqueous
solution of sodium alginate. In order to get the complete solution stirring is
continued and after that it was added drop wise to a solution containing Ca2+/Al3+
and chitosan solution in acetic acid. Microspheres which were formed were kept
in original solution for 24 hours for internal gellification followed by filtration for
separation.
ADVANTAGES:
• Controlled release delivery there by reducing side effects and eliminating the
inconvenience of repeated injections.
• Protein/peptide stability – microspheres helps to protect proteins because they are not
allowed to react with anything until the polymer is degraded, thus minimizing the
contact with solutions that could cause the proteins to react.
Ex: albumin prototype and lyzozymes.
• Drug targeting – it is the greatest advantage. Most drugs are targeted in the body to
give desired results either in specific tissues or organs.
Ex: It could be employed in targeting cancer cells in chemotherapy, as drugs and
chemical agents attack cancer cells but have a toxic effect on healthy ones which
could easily cause them to die.
• Gene delivery – Encapsulation of therapeutic agents such as DNA in microspheres
protects the agent from enzymatic degradation, enhances tissue specificity due to
localized delivery, eliminates the need for multiple administrations and allows for
sustained and controlled delivery.
• Microspheres are used with Gamma emitters such as Tc99 and 1131 for diagnostic
purposes.
APPLICATIONS:
• Microspheres in vaccine delivery: The prerequisite of a vaccine is protection against
the microorganism or its toxic product. An ideal vaccine must fulfil the requirement
of efficacy, safety, convenience in application and cost. Biodegradable delivery
systems for vaccines that are given by parenteral route may overcome the
shortcoming of the conventional vaccines.
• Targeting using micro particulate carriers: The therapeutic efficacy of the drug
relies on its access and specific interaction with its candidate receptors. Placement of
the particles in discrete anatomical compartment leads to their retention either because
of the physical properties of the environment or biophysical interaction of the
particles with the cellular content of the target tissue.
5. • Monoclonal antibodies mediated microspheres targeting: Monoclonal antibodies
targeting microspheres are immune-microspheres. This targeting is a method used to
achieve selective targeting to the specific sites. Monoclonal antibodies are extremely
specific molecules. This extreme specificity of monoclonal antibodies (Mabs) can be
utilized to target microspheres loaded bioactive molecules to selected sites. The Mabs
can be attached to microspheres by any of the following methods
1. Non specific adsorption
2. Specific adsorption
3. Direct coupling
4. Coupling via reagents
• Chemoembolisation: Chemoembolisation is an endovascular therapy, which involves
the selective arterial embolisation of a tumor together with simultaneous or
subsequent local delivery to chemotherapeutic agent. The theoretical advantage is that
such embolisations will not only provide vascular occlusion but will bring about
sustained therapeutic levels of chemotherapeutics in the areas of tumor.
• Imaging: The microspheres have been extensively studied and used for the targeting
purposes. Various cells, cell lines, tissues and organs can be imaged using radio
labelled microspheres. The particle size range of microspheres is an important factor
in determining the imaging of particular sites.
• Topical porous microspheres: Micro sponges are porous microspheres having
myriad of interconnected voids of particle size range 5-300 μm. These micro sponges
having capacity to entrap wide range of active ingredients such as emollients,
fragrances, essential oils etc., are used as the topical carrier system further, these
porous microspheres with active ingredients can be incorporated into formulations
such as creams, lotions and powders.
• Surface modified microspheres: Different approaches have been utilized to change
the surface properties of carriers to protect them against phagocytic clearance and to
alter their body distribution patterns. The adsorption of the poloxamer on the surface
of the polystyrene, polyester or poly methacrylate microspheres renders them more
hydrophilic and hence decreases their MPS uptake.
6. AIM:
To formulate Diclofenac micro-bead using two different polymeric systems:
(1) Sodium alginate microspheres using ionotropic gelation technique
(2) Ethyl cellulose microcapsules using solvent evaporation technique
REQUIREMENTS:
Chemicals required:
1) Polymers: Sodium alginate, Ethyl cellulose
2) Drug: Diclofenac
3) Solvent system: Isopropyl alcohol, Dichloro-methane, Distilled water
4) Surfactant: Tween 80
Equipments required:
Magnetic stirrer, 18-gauge hypodermic needle, 10 ml glass syringe, filters, hot air oven,
desiccators, beakers, glass rods etc.
FORMULATION:
Table 1: Formulation 1 (Sodium Alginate microbeads)
MATERIALS QUANTITY
Diclofenac (g) 0.1
Sodium alginate (g) 0.8
Water (ml) Q.S
Calcium chloride Q.S to make 4% w/w solution
Table 2: Formulation 2 (Ethyl cellulose microspheres)
MATERIALS F1 F2 F3
Diclofenac (g) 0.1 0.2 0.4
Ethyl cellulose (g) 0.8 0.7 0.5
PVP (g) 0.1 0.1 0.1
Dichloromethane & Isopropanol (1:1) 30 ml 30 ml 30 ml
Water 250 ml 250 ml 250 ml
Tween 80 1 ml 1 ml 1 ml
PROCEDURE:
1) Preparation of Diclofenac loaded Sodium Alginate micro-beads:
• The micro beads were prepared by ionotropic external gelation technique.
7. • Sodium alginate was dissolved in water using gentle heat and magnetic
stirring.
• On complete solution, an accurately weighed quantity of Diclofenac sodium
added and dispersed uniformly.
• The dispersion was sonicated for 30 min to remove any air bubbles formed
during the stirring process.
• The bubble free sodium alginate-drug dispersion (50ml) were added drop wise
via hypodermic needle into a mixture of 4% solution of Calcium chloride in
water & stirred at 500-1000 rpm for 30 min.
• The droplets from the dispersion gelled into discrete matrices upon contact
with the solution of gelling agent.
• The formed drug loaded micro-beads were stirred in solution of gelling agent
for an additional 1 hour.
• After specified time & stirring speed the gelled beads separated by filtration,
washed with H2O, dried at 60oC for 2 hours in hot air oven.
2) Preparation of Diclofenac loaded Ethyl cellulose microspheres:
• The micro-beads were prepared by double emulsion solvent evaporation
technique.
• Diclofenac was weighed, blended with Ethyl cellulose represented as F1, F2
and F3.
• The blend is added to the mixture of Isopropyl alcohol and dichloromethane
taken in the ratio 1:1.
• The dispersion was sonicated for 30 min to remove any air bubbles that may
formed during the stirring process.
• The above solution is mixed, added drop wise through hypodermic needle into
250 ml beaker of water (40oC) containing 1% tween and stirred at 500-1000
rpm for 30 min.
• The microspheres formed were stirred further for 0.5 – 3 hours.
• The entire solvent is allowed to evaporate & microspheres formed are
collected and stored.
EVALUATION OF MICROSPHERS
1. Assay:
• Diclofenac equivalent to 10 mg was weighed, transferred into a glass
mortar and crushed.
• To this 10-15 ml of methanol was added and transferred into the 100 ml
volumetric flask.
• Mortar is rinsed and the absorbance was checked at 274 nm on UV visible
spectrophotometer.
• The % assay was calculated from the absorbance of a standard drug
solution similarly prepared and diluted.
8. 2. In-vitro dissolution studies:
The in-vitro release of Diclofenac from microspheres was measured in phosphate
buffer medium (pH 7.4) by using UV spectrometer. Microspheres equivalent to
100 mg of the drug was taken into basket and the dissolution was performed for 2
hours. 5 ml of the sample was withdrawn for every 15 min, filtered and replaced
with fresh medium in order to maintain the sink condition. Suitable dilutions were
made and the absorbance was measured at 274 nm.
9. Table 3: Calibration curve of Diclofenac in 7.5 pH buffer at 275 nm
Concentation
(μg/ml)
Absorbance
2 0.057
4 0.106
6 0.151
8 0.185
10 0.218
Figure 1: Calibration curve of Diclofenac in 7.5 pH buffer at 275 nm
y = 0.020x + 0.023
0.25
0.2
0.15
0.1
0.05
Evaluation of microbeads (Sodium Alginate beads)
Table 4: Assay
Evaluation
parameter
Sodium
alginate beads
Diclofenac loaded Ethyl cellulose microsphere
F1 F2 F3
Assay (%w/w) 92.5
R² = 0.991
0
0 2 4 6 8 10 12
Series1
Linear (Series1)
