INTRODUCTION:

Microspheres can be defined as solid, approximately spherical particles ranging in size from 1 to 1000 μm. They are made of polymeric, waxy or other protective materials, which are biodegradable synthetic polymers and modified natural products such as starches, gums, proteins, fats and waxes. The natural polymers include albumin and gelatine, the synthetic polymer include poly lactic acid and polyglycolic acid. The solvents used to dissolve the polymeric materials chosen according to the polymer and drug solubility’s and stabilities, process safety and economic considerations. Microspheres are small and have large surface-to-volume ratio. At the lower end of their size range they have colloidal properties. The interfacial properties of microspheres are extremely important, often indicating their activity. ^{[1, 2]} Microspheres are characteristically free flowing powders consisting of proteins or polymers and ideally having a particle size less than 200 μm.

Due to its small particle size, are widely distributed throughout the gastrointestinal tract which improves drug absorption and reduces side effects due to localized build-up of irritating drugs against the gastrointestinal mucosa. ^[3]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. ^[4]

Advantages:

Ø Better process ability (improving solubility, dispersibility, flow ability)

Ø Self-life enhancement by preventing degradative reactions.

Ø Safe and convenient handling of toxic materials.

Ø Masking of odour or taste.

Ø Enzyme and microorganism immobilization.

Ø Controlled and targeted drug delivery.

Ø Handling liquids as solids.

Ø To improve bioavailability.

Ø To improve the stability.

Ø Limiting fluctuation within therapeutic range. ^{[ 5]}

Disadvantages:

Ø They are cleared and taken up from the circulation by reticuloendothelial cells.

Ø Premature drug release is seen.

Ø Poor entrapment of drug is seen.

Types of microspheres:

Bio adhesive microspheres:

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. ^[4]

Magnetic microspheres:

Magnetic carriers receive magnetic responses to a magnetic field from incorporated materials magnetic microspheres: Are used to deliver chemotherapeutic agent to liver tumour. ^[3]

Floating microspheres:

In floating types the bulk density is less than the gastric fluid and so remains buoyant in stomach. The drug is released slowly at the desired rate Moreover it also reduces chances of striking and dose dumping. One another way it produces prolonged therapeutic effect and therefore reduces dosing frequencies. Drug (ketoprofen) given through this form.^[6]

Radioactive microspheres:

They are injected to the arteries that lead totumors of interest. In these conditions radioactive microspheres deliver high radiation dose to the targeted areas without damaging the normal surrounding tissues, the different kinds of radioactive microspheres are α emitters, β emitters, γ emitters. ^[7]

Biodegradable polymeric microspheres:

Natural polymers prolongs the residence time when contact with aqueous medium, results gel formation. The rate and extent of drug release is controlled by concentration of polymer. The main drawback is, in clinical use drug loading efficiency of biodegradable microspheres is complex and is difficult to control the drug release. ^[7]

Synthetic polymeric microspheres:

The synthetic polymeric microspheres are widely used in clinical application and proved to be safe and biocompatible.⁵ But the main disadvantage of these kind of microspheres, are tend to migrate away from injection site and lead to potential risk, embolism and further organ damage. ^[8]

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. ^[9]

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 (eudragit) 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 hrs. ^[10]

Emulsion cross linking method:

In this method drug was dissolved in aqueous gelatine solution which was previously heated for 1 hr at 40 0C. The solution was added drop wise to liquid paraffin while stirring the mixture at 1500 rpm for 10 min at 35 oC, results in w/o emulsion then further stirring is done for 10 min at 15⁰C. Thus the produced microspheres were washed respectively three times with acetone and isopropyl alcohol which then air dried and dispersed in 5mL of aqueous glutaraldehyde saturated toluene solution at room temperature for 3 hrs for cross linking and then was treated with 100mL of 10mm glycine solution containing 0.1%w/v of tween 80 at 37⁰ C for 10 min to block unreacted glutaraldehyde.18 Examples for this technique is Gelatine A microspheres. ^[10]

Single emulsion technique:

The micro particulate carriers of natural polymers, i.e. those of proteins and carbohydrates are prepared by single emulsion technique. The natural polymers are dissolved/ dispersed in aqueous medium followed by dispersion in the non aqueous medium. Ex: oil. In the 2nd step, cross linking of the dispersed globule is carried out either by means of heat or by using chemical cross linkers. The chemical cross linking agents used –gluteraldehyde, formaldehyde, terephthalate chloride, diacidchloride, etc. Crosslinking by heat is affected by adding the dispersion to previously heated oil. Heat denaturation is not suitable for the thermo labile drugs while the chemical cross-linking suffers disadvantage of excessive exposure of active ingredient to chemicals if added at the time of preparation (fig.-1). ^[11]

Double emulsion technique:

Involves the formation of the multiple emulsions or the double emulsion of type w/o/w & is best suited to the water soluble drugs, peptides, proteins & the vaccines. The aqueous protein solution is dispersed in a lipophilic organic continuous phase which is generally consisted of polymer solution that eventually encapsulates protein contained in dispersed aqueous phase. The primary emulsion is then subjected to the homogenization before addition to aqueous solution of PVA .this results in formation of double emulsion which is then subjected to solvent removal by solvent evaporation maintaining the emulsion at reduced pressure or by stirring so that organic phase evaporates out (fig.-2). ^[12]

Fig: 1 Processing scheme for microspheres-preparation by single emulsion technique

Fig: 2 processing scheme for microspheres-preparation by double emulsion technique

Spray congealing:

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 cold air. The atomization leads to the formation of 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. ^[13]

Applications of Microspheres:

1 Microspheres in vaccine delivery:

The prerequisite of a vaccine is protection against the microorganism or its toxic product. Biodegradable delivery systems for vaccines that are given by parenteral route may overcome the shortcoming of the conventional vaccines. ^[14]

Topical porous microspheres:

These micro sponges having capacity to entrap wide range of active ingredients such as emollients, fragrances, essential oils etc., are used as the topical carries system further, these porous microspheres with active ingredients can be incorporated into formulations such as creams, lotions and powders. ^[15]

Microspheres in chemotherapy:

The most promising application of microspheres are possible to used as carriers for anti- tumour agents. Enhanced endocytic activity and leaky vasculature administrated microspheres. Stealth microspheres are prepared by coating with soluble polyoxy ethylene. The accumulation of non-stealth microspheres in Reticulo Endothelial System (RES) may also be exploited for cancer chemotherapy. ^[16-18]

Microspheres for DNA Delivery:

Microspheres have been recently used as a delivery vehicle for the transfer of plasmid DNA which leads to improve the transfer of plasmid DNA and their stability in the bio- environment38. Truong-Le & Co workers (1998) developed a novel system for gene delivery based on the use of DNA-gelatin microspheres/ nanoparticles formed by salt induced complex coacervation of gelatin & plasmid DNA. ^[19]

Fluorescent microspheres:

These are made up of polystyrene or poly vinyl toluene, mono disperse system ranging in size from 20nm to 4μm.Preparation of fluorescent microspheres comprising, swelling the polymeric microsphere so that fluorescent dyes may enter the microsphere pores. Unswellingthe polymeric microspheres so that the fluorescent dyes become physically entrapped in the pores. ^[20]

Microspheres for Ocular delivery:

The most applications of drug loaded ophthalmic delivery systems are for glaucoma therapy, especially cholinergic agonists like pilocarpine16. The short elimation half life of aqueous eye drops can be extended from a very short time (1-3 min) to prolonged time (15-20 min) using microspheres which have biodegradable properties e.g.Poly alkyl cyanoacrylate. ^[21]

Brain Tumour:

A microsphere-based system has been developed to deliver therapeutic agents to brain tumours. The polymer, poly(methylidene malonate), has been used to prepare 5-fluorouracil-sustained release biodegradable microspheres, in order to treat malignant brain tumours by local delivery of anti-neoplastic agents, This polymer presents a slow degradation rate, thus leading to a long-term local delivery system. ^[22]

New application:

Ø Assay - Coated microspheres provide measuring tool in biology and drug research

Ø Buoyancy - Hollow microspheres are used to decrease material density in plastics (glass and polymer)

Ø Ceramics - Used to create porous ceramics used for filters (microspheres melt out during firing, Polyethylene Microspheres)

Ø Cosmetics - Opaque microspheres used to hide wrinkles and give colour, Clear microspheres provide "smooth ball bearing" texture during application (Polyethylene Microspheres).

Ø Drug delivery - As miniature time release drug capsule made of, for example, polymers. A similar use is as outer shells of microbubble contrast agents used in contrast-enhanced ultrasound.

Ø Electronic paper - Dual Functional microspheres used in Gyricon electronic paper

Ø Personal Care - Added to Scrubs as an exfoliating agent (Polyethylene Microspheres)

Ø Spacers - Used in LCD screens to provide a precision spacing between glass panels (glass)

Ø Standards - monodispere microspheres are used to calibrate particle sieves, and particle counting apparatus.

Ø Retroreflective - added on top of paint used on roads and signs to increase night visibility of road stripes and signs (glass).

Ø Thickening Agent - Added to paints and epoxies to modify viscosity and buoyancy

Recent advances:

Wound healing properties:

Efficacy of chitosan in the promotion of wound healing was first reported in 1978.. Chitosan acetate films, which were tough and protective, had the advantage of good oxygen permeability, high water absorptivity and slow enzymatic degradation. ^[23]

Important utilizations of chitosan polymer:

Cholesterol-lowering effects Chitosan and cellulose were used as examples of fibers with high, intermediate and low bile acid-binding capacities, respectively. The serum cholesterol levels in a control group of mice fed a high fat/high cholesterol diet for 3 weeks increased about 2-fold to 4·3mM and inclusion of any of these fibers at 7·5% of the diet prevented this increase from occurring. In addition, the amount of cholesterol accumulated in hepatic stores due to the HFHC diet was reduced by treatment with these fibers. The three kinds of fibers showed similar hypocholesterolaemic activity; however, cholesterol depletion of liver tissue was greatest with cholestyramine.

The mechanisms underlying the cholesterol lowering effect of cholestyramine were,

1) Decreased cholesterol (food) intake,

2) Decreased cholesterol absorption efficiency, and

3) Increased faecal bile acid and cholesterol excretion. The latter effects can be attributed to the high bile acid-binding capacity of cholestyramine. In contrast, incorporation of chitosan or cellulose in the diet reduced cholesterol (food) intake, but did not affect either intestinal cholesterol absorption or faecal sterol output. The present study provides strong evidence that above all satiation and satiety effects underlie the cholesterol lowering. ^[23]

DNA Encapsulation:

Gene therapy holds tremendous potential for treating genetic diseases and acquired diseases including cancer, and as vaccines. A major barrier to development of gene-based pharmaceuticals is safe and efficient DNA delivery. Much research has focused on development of gene delivery vectors including viruses, liposome, and polymers. However, parenteral administration of naked plasmid DNA (pDNA) leads to gene expression, and controlled release of pDNA from polymeric matrices, micro particle and nanoparticles has been reported recently. In particular, encapsulation and controlled release of pDNA from biodegradable microspheres may provide a number of advantages including protection from nuclease degradation, access to alternative routes of administration (e.g., nasal, pulmonary, oral, and mucosal), passive targeting to professional antigen-presenting cells, and prolonged gene expression. ^[24]

Sterilization of microspheres:

Microspheres that are administered parenterally must be sterile. Sterilization is usually achieved by aseptic processing. The final product may not be able to undergo terminal sterilization, which may be detrimental to the delivery system, altering the release pattern or destroying the targeting properties.Sterility assurance is also a problem for microsphere system: although the exterior can be investigated for sterility by conventional plating methodology, it is difficult to determine whether the interiors of the microspheres are free from contamination. A method has been developed whereby the presence of viable organisms in the interior of microspheres systems can be determined without breaking the microcapsules/microspheres; it involves the detection of the organism metabolism. ^[25]

Characterization/ Evaluation of microspheres:

Particle size analyser:

Microsphere (50 mg) was suspended in distilled water (5mL) containing 2%w/v of tween 80, to prevent microsphere aggregation, the above suspension is sonicated in water bath and the particle size was expressed as volume mean diameter in micrometer. ^[26]

Optical microscopy:

This method was used to determine particle size by using optical microscope (Meizer OPTIK) The measurement was done under 450x (10x eye piece and 45x objective) and100 particles were calculated. ^[27]

Entrapment efficiency:

Microspheres containing of drug (5mg) were crushed and then dissolved in distilled water with the help of ultrasonic stirrer for 3 hr, and was filtered then assayed by uv-vis spectroscopy. Entrapment efficiency is equal to ratio of actual drug content to theoretical drug content. ^[28]

Stability studies:

By placing the microspheres in screw capped glass container and stored them at following conditions:

1. Ambient humid condition

2. Room temperature (27+/-2 0C)

3. Oven temperature (40+/-2 0C)

4. Refrigerator (5 0C -80C).

It was carried out of a 60 days and the drug content of the microsphere was analysed. ^[29]

% yield of microspheres:

Thoroughly dried microspheres were collected and weighed accurately. The percentage yield was then calculated using formula given below. ^[30-34]

% Yield = mass of microsphere obtained / total weight of drug & polymer X 100

Angle of repose:

Angle of repose was determined by using funnel method. The accurately weighed microspheres were taken in a funnel and then height of funnel was adjusted in such as way that the tip of funnel just touches the apex of heap of blends. The blends were allowed to flow through funnel freely on to surface. The diameter of powder cone was measured and angle of repose was calculated by using following equation. ^[30-34]

Tan q = h/r

Where

q –Angle of repose,

h –height of pile,

r – Radius of base.

Swelling studies:

A known weight (50 mg) of microspheres was placed in a glass vial containing 10 ml of distilled water at 37 ± 0.50C in incubator with occasional shaking. The microspheres were periodically removed, blotted with filter paper and their changes in weights were measured during the swelling until equilibrium was attained. Finally, the weight of the swollen microspheres was recorded after a period of 3 hours, and the swelling ratio (SR) was then calculated from the following formula. The studies were carried out in triplicate. ^[31-35]

SR=We-Wo / Wo

Where,

Wo = Initial weight of the dry microspheres,

We = Weight of the swollen microspheres at equilibrium swelling in the media.

Future challenges:

Future challenges of microspheres look bright particularly in the area of medicinal field because of its wide spectrum of application in molecular biology, e.g. microsphere based genotyping platform is used to detect six single nucleotide polymorphism, yittrium-90 microspheres is used to prevent tumour after liver transplantation and it’s advanced way in delivery of vaccines and proteins. Microspheres offer a unique carrier system for many cancer drugs and can be tailored to adhere to any cancerous tissue, including those found throughout the respiratory, urinary, and gastrointestinal tract. The microspheres can be used not only for controlled release, but also for targeted delivery of the drugs to specific sites in the body. Recent advances in medicine have envisaged the development of polymeric drug delivery systems for protein / peptide drugs and gene therapy. Although significant advances have been made in the field of microspheres, there are still many challenges ahead in this field. The most significant are the development of the universally acceptable standard evaluation methods and development of newer site-directed polymers. Polymeric science needs to be explored, to find newer microsphere polymers, with added attributes of being biodegradable, biocompatible, and bio adhesive for specific cells or mucosa, and which can also function as enzyme inhibitors for the successful delivery of proteins and peptides. A multidisciplinary approach will therefore be required to overcome these challenges and to employ microspheres as a cutting edge technology for site-targeted, controlled release drug delivery of new as well as existing drugs. The future direction of microspheres lies in potent and various other vaccine formulations that adhere to tissues and result in immunity. There is a need to look forward to further improvements in the formulations and drug delivery by these mechanisms, giving better tools to care for patients.

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Received on 19.01.2013 Modified on 03.02.2013

Research J. Pharm. and Tech. 6(3): March 2013; Page 307-312