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 obtain 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 effects1. 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 protein or synthetic polymers which are biodegradable in nature and ideally having a particle size less than 200 μm. In contrast to drug delivery system, the word novel is searching something out of necessity. The drug has to be delivered for a prolonged period of time and many medicines have to be taken simultaneously in case of chronic patients. Frequent administration of drug is necessary when those have shorter half life and all these leads to decrease in patient’s compliance. In order to overcome the above problems, various types of controlled release dosage forms are formulated and altered, so that patient compliance increase through prolonged effect, adverse effect decreases by lowering peak plasma concentration. The controlled release dosage form maintaining relatively constant drug level in the plasma by releasing the drug at a predetermined rate for an extended period of time. One such in Microspheres as carriers of drug become an approach of controlled release dosage form in novel drug delivery system. 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 or macroscopic level. It has a particle size of (1-1000nm).

Materials Used ⁽¹⁾

Microspheres used usually are polymers. They are classified into two types.

1. Synthetic Polymers

2. Natural polymers

Synthetic polymers are divided into two types.

i. Non-biodegradable polymers

· Poly methyl methacrylate (PMMA)

· Acrolein

· Glycidyl methacrylate

· Epoxy polymers

ii. Biodegradable polymers ^(1,2)

· Lactides, Glycolides and their co polymers

· Poly alkyl cyano Acrylates

· Poly anhydrides

Natural polymers obtained from different sources like proteins, carbohydrates and chemically modified carbohydrates. ^(3,4)

A] Proteins:

· Albumin

^·Gelatin ⁽⁵⁾

· Collagen

B] Carbohydrates:

· Agarose

· Carrageenan

^·Chitosan⁶

· Starch

C] Chemically modified carbohydrates:

^·Poly dextran⁷

· Poly starch.

TYPES OF MICROSPHERE ^(8,9,10)

1. Bioadhesive Microspheres ^(11,12,13)

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.

2. Magnetic Microspheres ⁽¹⁴⁾

This kind of delivery system is very much important which localizes 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 are chitosan, dextran etc. The different types are therapeutic magnetic microspheres and diagnostic microspheres.

i. Therapeutic Magnetic Microspheres: It is used to deliver chemotherapeutic agent to liver tumor. Drugs like proteins and peptides can also be targeted through this system.

ii. Diagnostic Microspheres: It can be used for imaging liver metastases and also can be used to distinguish bowel loops from other abdominal structures by forming nano size particles supramagnetic iron oxides.

3. Floating microspheres ^(15,16)

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 desired rate, if the system is floating on gastric content and increases gastric residence and increases fluctuation in plasma concentration. Moreover it also reduces chances of striking and dose dosing frequencies.

4. Polymeric Microspheres ⁽¹⁷⁾

The different types of polymeric microspheres can be classified as follows and they are biodegradable polymeric microspheres and synthetic polymeric microspheres.

i. Biodegradable Polymeric Microspheres:

Natural polymers such as starch are used with the concept that they are biodegradable, biocompatible, and also bioadhesive 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. The rate and extent of drug release is controlled by concentration of polymer and the release pattern in a sustained manner. The main drawback is in clinical use drug loading efficiency of biodegradable microspheres is complex and is difficult to control the drug release.

ii. 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.

ADVANTAGES

1. Microspheres provide constant and prolonged therapeutic effect.

2. Reduces the dosing frequency and thereby improve the patient compliance.

3. They could be injected into the body due to the spherical shape and smaller size.

4. Better drug utilization will improve the bioavailability and reduce the incidence or intensity of adverse effects.

5. Microsphere morphology allows a controllable variability in degradation and drug release⁽¹⁸⁾

LIMITATION

Some of the disadvantages were found to be as follows

1. The modified release from the formulations.

2. The release rate of the controlled release dosage form may vary from a variety of factors like food and the rate of transit though gut.

3. Differences in the release rate from one dose to another.

4. Controlled release formulations generally contain a higher drug load and thus any loss of integrity of the release characteristics of the dosage form may lead to potential toxicity.

5. Dosage forms of this kind should not be crushed or chewed.¹⁸

CHARACTERISTICS OF MICROSPHERES:

Table 1: Microsphere property ⁽¹⁹⁾

S. No	Property	Consideration
1	Size Diameter	Uniformity/distribution
2	Composition	Density, Refractive Index, Hydrophobicity/ hydrophilicity Nonspecific binding Autofluorescence
3	Surface Chemistry	Reactive groups Level of functionalization Charge
4	Special Properties	Visible dye/ fluorophore Superparamagnetic

1. Microsphere size may be critical to the proper function of an assay, or it may be secondary to other characteristics. Considering traditional diagnostic methods, the test or assay format commonly dictates particle size, such as the use of very small spheres (~0.1- 0.4μm) to ensure satisfactory wicking in lateral flow tests, or the use of larger, cell-sized spheres (~4 10μm) for bead based flow cytometric assays.

2. Common microsphere compositions include polystyrene (PS), poly(methyl methacrylate) (PMMA), and silica. These materials possess different physical and optical properties, which may present advantages or limitations for different applications. Polymer beads are generally hydrophobic, and as such, have high protein binding abilities. However, they often require the use of some surfactant (e.g. 0.01-0.1% Tween® 20 or SDS) in the storage buffer to ensure ease of handling. During synthesis, functional monomers may be co-polymerized with styrene or methyl methacrylate to develop beads with surface reactive groups. Functional groups may be used in covalent binding reactions, and also aid in stabilizing the suspension. Silica microspheres are inherently hydrophilic and negatively charged. Consequently, aqueous silica suspensions rarely require use of surfactants or other stabilizers. Carboxyl- and amine functionalized silica spheres are available for use in common covalent coating protocols, and plain silica microspheres may be modified using a variety of silanes to generate functional groups or alter surface properties.

3. Microspheres may be coated with capture molecules, such as antibodies, oligonucleotides, peptides, etc. for use in diagnostic or separation applications. Microsphere coatings are typically optimized to achieve desired specific activity, while minimizing nonspecific interactions. Consideration should also be given to the required stability, development time frame and budget, and the specific biomolecule to be coated. These factors will aid in determining the most fitting coating strategy for both short- and long-term objectives. Standard microsphere products support three basic coating strategies: adsorption, covalent coupling, and affinity binding.

4. Many applications in the life sciences demand added properties, such as fluorescence or a visible color, or iron oxide inclusions for magnetic separations. Polymer spheres (and polymer based magnetic spheres) are often internally dyed via organic solvent swelling, and many standard products are available. Dye concentrations can be adjusted to produce beads with different intensities to meet special needs, such as Quantum Plex™ for multiplexed flow cytometric assays, or our Dragon Green or Flash Red Intensity Standards, which support imaging applications and associated instrument QC. Many surface- or internally labeled fluorescent beads are also available as specialized

flow cytometry standards⁽²⁴⁾

CRITERIA FOR MICROSPHERE PREPARATION:

Incorporation of solid, liquid or gases into one or more polymeric coatings can be done by micro encapsulation technique ⁽²⁰⁾. The 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, coprecipitation etc ⁽²¹⁾. Preparation of microspheres should satisfy certain criteria ⁽²²⁾

1. The ability to incorporate reasonably high concentrations of the drug.

2. Stability of the preparation after synthesis with a clinically acceptable shelf life.

3. Controlled particle size and dispersability in aqueous vehicles for injection.

4. Release of active reagent with a good control over a wide time scale.

5. Biocompatibility with a controllable biodegradability and

6. Susceptibility to chemical modification.

METHOD OF PREPARATION

Spray Drying

In Spray Drying the polymer is first dissolved in a suitable volatile organic solvent such as dichloromethane, Acetone, etc. The drug in the solid form is then dispersed in the polymer solution under high-speed homogenization. This dispersion is then atomized in a stream of hot air. The atomization leads to the formation of the small droplets or the fine mist from which the solvent evaporate instantaneously leading the formation of the microspheres in a size range 1-100μm. Micro particles are separated from the hot air by means of the cyclone separator while the trace of solvent is removed by vacuum drying. One of the major advantages of process is feasibility of operation under aseptic conditions this process is rapid and this leads to the formation of porous micro particles

Solvent Evaporation

The processes are carried out in a liquid manufacturing vehicle. The microcapsule coating is dispersed in a volatile solvent which is immiscible with the liquid manufacturing vehicle phase. A core material to be microencapsulated is dissolved or dispersed in the coating polymer solution. With agitation the core material mixture is dispersed in the liquid manufacturing vehicle phase to obtain the appropriate size microcapsule. The mixture is then heated if necessary to evaporate the solvent for the polymer of the core material is disperse in the polymer solution, polymer shrinks around the core. If the core material is dissolved in the coating polymer solution, matrix – type microcapsules are formed. The core materials may be either water soluble or water in soluble materials. Solvent evaporation involves the formation of an emulsion between polymer solution and an immiscible continuous phase whether aqueous (o/w) or non aqueous. The comparison of mucoadhesive microspheres of hyaluronic acid, Chitosan glutamate and a combination of the two prepared by solvent evaporation with microcapsules of hyaluronic acid and gelating prepared by complex coacervation were made.

Wet Inversion Technique

Chitosan solution in acetic acid was dropped in to an aqueous solution of counter ion sodium tripolyposphate through a nozzle. Microspheres formed were allowed to stand for 1 hr and cross linked with 5% ethylene glycol diglysidyl ether. Microspheres were then washed and freeze dried. Changing the pH of the coagulation medium could modify the pore structure of CS microspheres.

Complex Coacervation

CS microparticles can also prepare by complex co acervation, Sodium alginate, sodium CMC and sodium polyacrylic acid can be used for complex coacervation with CS to form microspheres. These microparticles are formed by interionic interaction between oppositely charged polymers solutions and KCl and CaCl2 solutions. The obtained capsules were hardened in the counter ion solution before washing and drying ⁽²³⁾

Hot Melt Microencapsulation

The polymer is first melted and then mixed with solid particles of the drug that have been sieved to less than 50 μm. The mixture is suspended in a non-miscible solvent (like silicone oil), continuously stirred, and heated to 5°C above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decantation with petroleum ether. The primary objective for developing this method is to develop a microencapsulation process suitable for the water labile polymers, e.g. poly anhydrides. Microspheres with diameter of 1- 1000 μm can be obtained and the size distribution can be easily controlled by altering the stirring rate. The only disadvantage of this method is moderate temperature to which the drug is exposed ⁽²³⁾

Single emulsion technique

The micro particulate carriers of natural polymers of natural polymers i.e. those of proteins and carbohydrates are prepared by single emulsion technique. The natural polymers are dissolved or dispersed in aqueous medium followed by dispersion in non-aqueous medium like oil. Next cross linking of the dispersed globule is carried out. The cross linking can be achieved either by means of heat or by using the chemical cross linkers. The chemical cross linking agents used are glutaraldehyde, formaldehyde, acid chloride etc. Heat denaturation is ^not suitable for thermolabile substances. Chemical cross linking suffers the disadvantage of excessive exposure of active ingredient to chemicals if added at the time of preparation and then subjected to centrifugation, washing, separation ⁽²⁴ ⁾.The nature of the surfactants used to stabilize the emulsion phases can greatly influence the size, size distribution, surface morphology, loading, drug release, and bio performance of the final multiparticulate product.⁽²⁵⁾

Double emulsion technique

Double emulsion method of microspheres preparation involves the formation of the multiple emulsions or the double emulsion of type w/o/w and is best suited to water soluble drugs, peptides, proteins and the vaccines. This method can be used with both the natural as well as synthetic polymers. The aqueous protein solution is dispersed in a lipophilic organic continuous phase. This protein solution may contain the active constituents. The continuous phase is generally consisted of the polymer solution that eventually encapsulates of the protein contained in dispersed aqueous phase. The primary emulsion is subjected then to the homogenization or the sonication before addition to the aqueous solution of the poly vinyl alcohol (PVA). This results in the formation of a double emulsion. The emulsion is then subjected to solvent removal either by solvent evaporation or by solvent extraction. a number of hydrophilic drugs like leutinizing hormone releasing hormone (LH-RH) agonist, vaccines, proteins/peptides and conventional molecules are successfully incorporated into the microspheres using the method of double emulsion solvent evaporation/ extraction.

Polymerization techniques

The polymerization techniques conventionally used for the preparation of the microspheres

are mainly classified as:

I. Normal polymerization

II. Interfacial polymerization. Both are carried out in liquid phase.

Normal polymerization

It is carried out using different techniques as bulk, suspension, precipitation, emulsion and micellar polymerization processes. In bulk, a monomer or a mixture of monomers along with the initiator or catalyst is usually heated to initiate polymerization. Polymer so obtained may be moulded as microspheres. Drug loading may be done during the process of polymerization. Suspension polymerization also referred as bead or pearl polymerization. Here it is carried out by heating the monomer or mixture of monomers as droplets dispersion in a continuous aqueous phase. The droplets may also contain an initiator and other additives. Emulsion polymerization differs from suspension polymerization as due to the presence initiator in the aqueous phase, which later on diffuses to the surface of micelles. Bulk polymerization has an advantage of formation of pure polymers.

Interfacial polymerization

It involves the reaction of various monomers at the interface between the two immiscible

liquid phases to form a film of polymer that essentially envelops the dispersed phase.

Phase separation coacervation technique

This process is based on the principle of decreasing the solubility of the polymer in organic phase to affect the formation of polymer rich phase called the coacervates. In this method, the drug particles are dispersed in a solution of the polymer and an incompatible polymer is added to the system which makes first polymer to phase separate and engulf the drug particles. Addition of non-solvent results in the solidification of polymer. Poly lactic acid (PLA) microspheres have been prepared by this method by using butadiene as incompatible polymer. The process variables are very important since the rate of achieving the coacervates determines the distribution of the polymer film, the particle size and agglomeration of the formed particles. The agglomeration must be avoided by stirring the suspension using a suitable speed stirrer since as the process of microspheres formation begins the formed polymerize globules start to stick and form the agglomerates. Therefore the process variables are critical as they control the kinetic of the formed particles since there is no defined state of equilibrium attainment.

Spray drying and spray congealing

These methods are based on the drying of the mist of the polymer and drug in the air. Depending upon the removal of the solvent or cooling of the solution, the two processes are named spray drying and spray congealing respectively. The polymer is first dissolved in a suitable volatile organic solvent such as dichloromethane, acetone, etc. The drug in the solid is then dispersed in the polymer solution under high speed homogenization. This dispersion is then atomized in a stream of hot air. The atomization leads to the formationof the small droplets or the fine mist from which the solvent evaporates instantaneously leading the formation of the microspheres in a size range 1-100 μm. Microparticles are separated from the hot air by means of the cyclone separator while the traces of solvent are removed by vacuum drying. One of the major advantages of the process is feasibility of operation under aseptic conditions. The spray drying process is used to encapsulate various penicillins. Thiamine mononitrate and sulpha ethylthiadizole are encapsulated in a mixture of mono- and diglycerides of stearic acid and palmiticacid using spray congealing. Very rapid solvent evaporation, however leads to the formation of porous microparticles.

Solvent extraction

Solvent evaporation method is used for the preparation of microparticles, involves removal of the organic phase by extraction of the organic solvent. The method involves water miscible organic solvents such as isopropanol. Organic phase is removed by extraction with water. This process decreases the hardening time for then microspheres. One variation of the process involve direct addition of the drug or protein to polymer organic solution. The rate of solvent removal by extraction method depends on the temperature of water, ratio of emulsion volume to the water and the solubility profile of the polymer ⁽²⁴⁾

Fig.1. Solvent Extraction Method ⁽²⁶⁾

Preparation of Microspheres by Thermal cross-linking

Citric acid, as a cross-linking agent was added to 30 mL of an aqueous acetic acid solution of chitosan (2.5% wt/vol) maintaining a constant molar ratio between chitosan and citric acid (6.90 × 10−3 mol chitosan: 1 mol citric acid). The chitosan cross-linker solution was cooled to 0°C and then added to 25 mL of corn oil previously maintained at 0°C, with stirring for 2 minutes. This emulsion was then added to 175 mL of corn oil maintained at 120°C, and cross-linking was performed in a glass beaker under vigorous stirring (1000 rpm) for 40 minutes. The microspheres obtained were filtered and then washed with diethyl ether, dried, and sieved ⁽²⁷⁾

Preparation of Microspheres by Glutaraldehyde cross linking

A 2.5% (w/v) chitosan solution in aqueous acetic acid was prepared. This dispersed phase was added to continuous phase (125 mL) consisting of light liquid paraffin and heavy liquid paraffin in the ratio of 1:1 containing 0.5% (wt/vol) Span 85 to form a water in oil (w/o) emulsion. Stirring was continued at 2000 rpm using a 3- blade propeller stirrer). A drop-by-drop solution of a measured quantity (2.5 mL each) of aqueous glutaraldehyde (25% v/v) was added at 15, 30, 45, and 60 minutes. Stirring was continued for 2.5 hours and separated by filtration under vacuum and washed, first with petroleum ether (60°C-80°C) and then with distilled water to remove the adhered liquid paraffin and glutaraldehyde, respectively. The microspheres were then finally dried in vacuum desiccators ⁽²⁸⁾

Physicochemical Evaluation:

i) Characterization

The characterization of the microparticulate carrier is an important phenomenon, which helps to design a suitable carrier for the proteins, drug or antigen delivery. These microspheres have different microstructures. These microstructures determine the release and the stability of the carrier ⁽²⁹⁾.

Particle Size and Shape

The most widely used procedures to visualize microparticles are conventional light microscopy (LM) and scanning electron microscopy (SEM). Both can be used to determine the shape and outer structure of microparticles. LM provides a control over coating parameters in case of double walled microspheres. The microspheres structures can be visualized before and after coating and the change can be measured microscopically. SEM provides higher resolution in contrast to the LM ⁽³⁰⁾.SEM allows investigations of the microspheres surfaces and after particles are cross-sectioned, it can also be used for the investigation of double walled systems. Conflocal fluorescence microscopy1 is used for the structure characterization of multiple walled microspheres. Laser light scattering and multi size coulter counter other than instrumental methods, which can be used for the characterization of size, shape and morphology of the microspheres.

Electron Spectroscopy for Chemical Analysis:

The surface chemistry of the microspheres can be determined using the electron spectroscopy for chemical analysis (ESCA). ESCA provides a means for the determination of the atomic composition of the surface. The spectra obtained using ECSA can be used to determine the surfacial degradation of the biodegradable microspheres.

Attenuated total reflectance Fourier Transfom-

Infrared Spectroscopy:

FT-IR is used to determine the degradation of the polymeric matrix of the carrier system. The surface of the microspheres is investigated measuring alternated total reflectance (ATR). The IR beam passing through the ATR cell reflected many times through the sample to provide IR spectra mainly of surface material. The ATRFTIR provides information about the surface composition of the microspheres depending upon manufacturing procedures and conditions.

Density Determination:

The density of the microspheres can be measured by using a multi volume pycnometer. Accurately weighed sample in a cup is placed into the multi volume pycnometer. Helium is introduced at a constant pressure in the chamber and allowed to expand. This expansion results in a decrease in pressure within the chamber. Two consecutive readings of reduction in pressure at different initial pressure are noted. From two pressure readings the volume and hence the density of the microsphere carrier is determined.

Isoelectric Point:

The micro electrophoresis is an apparatus used to measure the electrophoretic mobility of microspheres from which the isoelectric point can be determined. The mean velocity at different Ph values ranging from 3-10 is calculated by measuring the time of particle movement over a distance of 1 mm. By using this data the electrical mobility of the particle can be determined. The electrophoretic mobility can be related to surface contained charge, ionisable behaviour or ion absorption nature of the microspheres.

Surface Carboxylic Acid Residue:

The surface carboxylic acid residue is measured by using radioactive glycine. The radioactive glycine conjugates is prepared by the reaction of c14-glycine ethyl ester hydro chloride with the microspheres. The glycine residue is linked using the water soluble condensing 1- ethyl-3 (3-dimethyl amino propyl) carbidiimide (EDAC). The radioactivity of the conjugate is then measured using liquid scintillation counter. Thus the carboxylic acid residue can be compared and correlated. The free carboxylic acid residue can be measured for hydrophobic or hydrophilic or any other derivatized type of the microspheres.

Surface Amino Acid Residue:

Surface associated amino acid residue is determined by the radioactive c14-acetic acid conjugate. The carboxylic acid residue is measured through the liquid scintillation counter and hence the amino acid residue can be determined indirectly. EDAC is used to condense the amino group and the c14 –acetic acid carboxylic acid residue. The method used for determining the free amino or the free carboxylic acid residues are based on indirect estimation, by measuring the radioactivity of the c14 having acetic acid or the glycine conjugate. The accuracy of the method however, depends on the time allowed for conjugation of the radioactive moiety and the reactivity of free functional group.

Capture Efficiency:

The capture efficiency of the microspheres or the percent entrapment can be determined by allowing washed microspheres to lyse. The lysate is then subjected to the determination of active constituents as per monograph requirement. The percent encapsulation efficiency is calculated using following equation:

% Entrapment = Actual content/Theoretical content x 100

Angle of Contact:

The angle of contact is measured to determine the wetting property of a micro particulate carrier. It determines the nature of microspheres in terms of hydrophilicity or hydrophobicity. This thermodynamic property is specific to solid and affected by the presence of the adsorbed component. The angle of contact is measured at the solid/air/water interface. The advancing and receding angle of contact are measured by placing a droplet in a circular cell mounted above objective of inverted microscope. Contact angle is measured at 2000C within a minute of deposition of microspheres.

In - Vitro methods

There is a need for experimental methods which allow the release characteristics and permeability of a drug through membrane to be determined. For this purpose, a number of in vitro and in vivo techniques have been reported. In vitro drug release studies have been

employed as a quality control procedure in pharmaceutical production, in product development etc. Sensitive and reproducible release data derived from physico chemically and hydro dynamically defined conditions are necessary. The influence of technologically defined conditions and difficulty in simulating in vivo conditions has led to development of a number of in vitro release methods for buccal formulations; however no standard in vitro method has yet been developed. Different workers have used apparatus of varying designs and under varying conditions, depending on the shape and application of the dosage form developed ⁽³¹⁾

Beaker Method ^(32-35)

The dosage form in this method is made to adhere at the bottom of the beaker containing the medium and stirred uniformly using over head stirrer. Volume of the medium used in the literature for the studies varies from 50-500 ml and the stirrer speed form 60-300 rpm.

Interface Diffusion System

This method is developed by Dearden and Tomlinson. It consists of four compartments. The compartment A represents the oral cavity, and initially contained an appropriate concentration of drug in a buffer. The compartment B representing the buccal membrane, contained 1-octanol, and compartment C representing body fluids, contained 0.2 M HCl. The compartment D representing protein binding also contained 1-octanol. Before use, the aqueous phase and 1- octanol were saturated with each other. Samples were withdrawn and returned to compartment A with a syringe.

Modified Keshary Chien Cell ^(36,37)

A specialized apparatus was designed in the laboratory. It comprised of a Keshary Chien cell containing distilled water (50ml) at 370 C as dissolution medium. TMDDS (Trans Membrane Drug Delivery System) was placed in a glass tube fitted with a 10# sieve at the bottom which reciprocated in the medium at 30 strokes per min.

Dissolution Apparatus:

Standard USP or BP dissolution apparatus have been used to study in vitro release profiles using both rotating elements, paddle^(38-40) and basket^(41,42) .Dissolution medium used for the study varied from 100- 500 ml and speed of rotation from 50-100 rpm.

Other Methods:

Few other methods involving plexi glass sample blocks placed in flasks ⁽⁴³⁾ agar gel method ⁽⁴⁴⁾, Valia-Chein cell USP n2 III dissolution apparatus ^(45,46) etc have also been reported. Although a number of methods have been reported, the ideal method would be one where sink condition is maintained and dissolution time in vitro simulates dissolution time in vivo.

In -vivo methods

Methods for studying the permeability of intact mucosa comprise of techniques that exploit the biological response of the organism locally or systemically and those that involve direct local measurement of uptake or accumulation of penetrants at the surface. Some of the earliest and simple studies of mucosal permeability utilized the systemic pharmacological effects produced by drugs after application to the oral mucosa. However the most widely used methods include in vivo studies using animal models, buccal absorption tests, and perfusion chambers for studying drug permeability.⁽⁴⁷⁾

Animal Models

Animal models are used mainly for the screening of the series of compounds, investigating the mechanisms and usefulness of permeation enhancers or evaluating a set of formulations. A number of animal models have been reported in the literature, however, very few in vivo (animal). Animal models such as the dog ^(48,49), rats ⁽⁵⁰⁾, rabbits ^(51,52) cat ⁽⁵³⁾ ,hamster ^(54,55), pigs ⁽⁵⁶⁾, and sheep ⁽⁵⁷⁾ have been reported. In general, the procedure involves anesthetizing the animal followed by administration of the dosage form. In case of rats, the oesophagus is ligated to prevent absorption pathways other than oral mucosa. At different time intervals, the blood is withdrawn and analyzed.

In vitro-In vivo correlations

Correlations between in vitro dissolution rates and the rate and extent of availability as determined by blood concentration and or urinary excretion of drug or metabolites are referred to as “in vitro-in vivo correlations”⁽⁵⁸⁾. Such correlations allow one to develop product specifications with bioavailability.

Percent of Drug Dissolved In Vitro Vs Peak Plasma Concentration:

One of the ways of checking the in vitro and in vivo correlation is to measure the percent of the drug released from different dosage forms and also to estimate the peak plasma concentrations achieved by them and then to check the correlation between them. It is expected that a poorly formulated dosage form releases amount of drug than a well formulated dosage form, and, hence the amount of drug available for absorption is less for poorly formulated dosage form than from a well formulated dosage form.

Percent of Drug Dissolved Vs Percent of Drug Absorbed:

If the dissolution rate is the limiting step in the absorption of the drug, and is absorbed completely after dissolution, a linear correlation may be obtained by comparing the percent of the drug absorbed to the percent of the drug dissolved. If the rate limiting step in the bioavailability of the drug is the rate of absorption of the drug, a change in the dissolution rate may not be reflected in a change in the rate and the extent of drug absorption from the dosage form.

Dissolution Rate Vs Absorption Rate:

The absorption rate is usually more difficult to determine than the absorption time. Since the absorption rate and absorption time of a drug are inversely correlated, the absorption time may be used in correlating the dissolution data to the absorption data. In the analysis of in vitro and in vivo drug correlation, rapid drug absorption may be distinguished from the slower drug absorption by observation of the absorption time for the dosage form. The quicker the absorption of the drug the less is the absorption time required for the absorption of the certain amount of the drug. The time required for the absorption of the same amount of drug from the dosage form is correlated.

Percent of Drug Dissolved Vs Serum Drug Concentration:

For drugs whose absorption from GIT is dissolution rate limited, a linear correlation may be established between the percent of drug dissolved at specified times and the serum drug concentrations at corresponding times.

Percent of Drug Dissolved Vs Percent of the Dose Excreted in Urine:

The percent of a drug dissolved and the percent of drug absorbed are linearly correlated. There exists a correlation between the amount of drug in body and the amount of drug excreted in the urine. Therefore, a linear relation may be established between the percent of the drug dissolved and the percent of the dose excreted in the urine ⁽⁵⁹⁾.

Application of microspheres in pharmaceutical industry

· For Taste and odour masking

· To delay the volatilisation

· For Separation of incompatible substances

· For Improvement of flow properties of powders

· To Increase the stability of the drug against the external conditions

· For Safe handling of toxic substances

· To Improve the solubility of water insoluble substances by incorporating dispersion of such material in aqueous media

· To reduce the dose dumping potential compared to large implantable devices.

· For conversion of oils and other liquids to solids for ease of handling

Novel Applications of Microsphere

Monoclonal antibodies mediated microspheres targeting

Monoclonal antibodies (Mabs) targeting microspheres are immune microspheres. This targeting is a method used to achieve selective targeting at specific sites. Monoclonal antibodies are extremely specific molecules. This extreme specificity of monoclonal antibodies (Mabs) can be used to target microspheres loaded bioactive molecules to selected sites by means of covalent coupling. The free amino groups, aldehyde groups, or hydroxyl groups on the external surface of the microspheres can be linked to the antibodies. Attachment of microspheres to Mabs by any of the following methods

1. Non specific adsorption

2. Specific adsorption

3. Direct coupling

4. Coupling with reagents

Targeting by using microparticulate carriers

The concept of targeing, i.e. site specific drug delivery is a well established dogma, which is gaining full attention. The therapeutic efficacy of the drug depends on its access and specific interaction with its candidate receptors. Placement of the particles indiscrete anatomical compartment leads to their retention either due to the physical properties of the environment or biophysical interaction of the particles with the cellular content of the target tissue.

Microspheres in vaccine delivery^(60,61)

The prerequisite of a vaccine is protection against micro organism or its toxic product. An ideal vaccine must fulfill the requirement of efficacy, convenience in application and cost. The aspect of safety and minimization of side effect is a complex issue. Biodegradable delivery systems for vaccines that are given by I .V. route may overcome the shortcoming of the conventional vaccines. The interest in parenteral (subcutaneous, intramuscular, intradermal) carrier lies because they offer specific advantages including:

1. Modulation of antigen release

2. Improved antigenicity

3. Stabilization of antigen.

Topical porous microspheres

These microsponges are having capacity to entrap wide range of active ingredients such as emollients, fragrances, volatile oils etc., are used as the topical carries system furthermore, these porous microspheres with active medicaments can be incorporated into formulations such as creams, lotions and powders. .

Surface modified microspheres

microspheres deviate them more hydrophilic and hence they decrease their MPS uptake. Protein microspheres can be covalently modified by PEG derivatives show decreased immunogenicity and clearance.

REFERENCES:

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