Nature has chosen two different but related polysaccharides to provide structure and integrity to plants and animals like crustaceans and insects. Plants produce cellulose in their cell walls and insects and crustaceans produce chitin in their shells. Like other polysaccharides, chitin and cellulose constitute polysaccharide chains built up from monosugars. Cellulose molecules are large chains of glucose units while chitin molecules are large chains of N-acetyl glucosamine units. Since these materials are crucial structural components of many organisms widely spread throughout the biosphere, cellulose and chitin are two of the most abundant biopolimers on earth.^1–3. The success of chitosan as carriers or microspheres is due to the following features:

1. They can dissolve poorly soluble drugs and thus increase their bioavailability.

2. They can stay in the body (in the blood) long enough to provide gradual accumulation in the required area.

3. Their size permits them to accumulate in body regions with leaky vasculature.

4. They can be tailored to achieve targeting or other desired properties by attachment of a specific ligand to the outer surface.

5. They have low toxicity and a high loading capacity, as well as minimize drug degradation and loss

6. They can be easily produced in large quantities.^4,6

More importantly, chitosan can be chemically modified through its amino and/or hydroxyl groups to form complexes and introduce desired functionalities for specific purposes. Many studies have been conducted on the design and fabrication of chitosan-based hybrid systems in order to achieve improved mechanical properties as well as improved biological performances. Various sustained release drug carriers have been made from chitosan such as microparticles tablets, gel and beads.

Bioadhesive:

Bioadhesive Controlled Release Microspheres:

A tetracycline microsphere was prepared for maximum bioadhesivity and controlled drug release. A formulation comprising of 3% (w/w) chitosan, 10% (w/w) tetracycline HCl and 9% (w/v) tripolyphosphate was identified for maximizing bioadhesivity and obtaining controlled drug release. The optimal microsphere preparation was subsequently characterised in terms of hydration dynamics, release kinetics, antimicrobial activity, thermal properties, morphology and surface pH. Antimicrobial studies showed that the drug concentrations in the in vitro release samples were above the minimum concentration of drug required for inhibition of Staphylococcus aureus growth⁷.

As Carrier for drug delivery:

As a controlled release system for protein, peptide and vaccine and biotechnologically derived product chitosan shows advantages as follows:

1. Chitosan is soluble in a weak acidic solution, and the use of organic solvent can be avoided which is favorable for maintaining bioactivity of protein and peptide drug.

2. The amino groups of chitosan are protonated in an acidic solution and the resultant soluble polysaccharide is positively charged, which can bind strongly to negatively charged surface such as cell surface and mucosa. Therefore, chitosan formulation can greatly improve the residence time of drug on tissues and cells and release the drug sustainedly there, as a result, the bio-availability of drug can be improved, the administration frequency of drug can be reduced

3. As a vaccine delivery system,chitosan can stimulate immunity system and works as an adjuvant found that chitosan-based influenza antigen produced much higher antibody level than other polymer-based antigen system⁸.

As a carrier of protein drug serum albumin (BSA):

Chitosan microspheres were used as a carrier of protein drugs like Bovine serum albumin (BSA). The chitosan microspheres were prepared by a membrane emulsification technique. Chitosan was dissolved in 1 % aqueous acetic acid containing 0.9 wt. % sodium chloride, which was used as a water phase. A mixture of liquid paraffin and petroleum ether 7:5 (v/v) containing emulsifier was used as an oil phase. The water phase was permeated through the uniform pores of a porous glass membrane into the oil phase by the pressure of nitrogen gas to form W/O emulsion. Then GST (Glutaraldehyde Saturated Toluene) as crosslinking agent was slowly dropped into the W/O emulsion to solidify the chitosan droplets. The preparation condition for obtaining uniform-sized microspheres was optimized.

Microspheres for biotechnologically derived products:

New types of medical agents such as peptides and proteins coming out of the biotech industry were widely used in the pharmaceutical field in the last decade. Such substances require new types of release systems because peptides and proteins are unstable in a biological environment without necessary protection. The application of these molecules as therapeutic agents depends on the design of an appropriate controlled release system for the delivery of such peptides and proteins.

Chitosan is a positively charged polysaccharide. This property enables it to react with negatively charged polymers and polyanions such as heparin and hyaluronic acid by electrostatic forces. Here heparin was used as polyanions cross-linked with chitosan microspheres by complexing to strengthen the protein drug controlled release system.Chitosan/heparin microspheres were prepared using the water-in-oil emulsification solvent evaporation technique. The prepared chitosan/heparin microspheres may provide a useful controlled release protein drug system for used in pharmaceutics⁹.

Chitosan microspheres containing insulin or other proteins:

It is known that microencapsulation of labile proteins improves their protection against gastric pH and enzymatic attack, providing a controlled release profile of the entrapped molecules and further enhancement of their intestinal absorption. Microencapsulation of proteins can be performed using alginate, chitosan and dextran sulphate. Chitosan microsphere has potential applications in orally and other mucosally administration of protein and peptide drug, because it shows excellent mucoadhesive and permeation enhancing effect across the biological surfaces. The control of the size and size distribution of chitosan microsphere is necessary in order to improve its reproducibility, bioavailability and repeatable release behavior.

The chitosan aqueous solution containing insulin was permeated through the membrane pores into the oil phase to form the W/O emulsions with uniform size under adequate pressure, finally the droplets of emulsions were solidified by a drop-wise crosslinking method. The uniform size of protein-loaded microspheres was expected to increase bioavailability of protein drug^{10, 11}.

Mannose-bearing chitosan microspheres for gene delivery:

A novel approach involving the preparation of mannose-bearing chitosan microspheres with entrapping gene or DNA was developed to improve the delivery of DNA into antigen-presenting cells after intramuscular injection. Compared with the traditional chitosan microspheres, the microspheres could quickly release intact and penetrative gene or DNA. Chitosan was modified with mannose to target the primary antigen-presenting cells such as dendritic cells owing to the high density of mannose receptors expressing on the surface of immature dendritic cells. After i.m. immunization, the microspheres induced significantly enhanced serum antibody and cytotoxic T lymphocyte (CTL) responses in comparison to naked DNA¹².

Chitosan microspheres as carriers of LH-RH analogue TX46:

Two kinds of swellable chitosan microspheres were prepared by cross-linking technique and dry-in-oil method. The polypeptide drug Luteinizing hormone-releasing hormone (LH-RH) analogue (TX46 as a model drug) was loaded in the microspheres and released in vitro. A novel kind of chitosan microspheres, which contained glycine were also prepared and employed to adsorb and release TX46.¹³.

Monodisperse tripolyphosphate (TPP)-chitosan microparticles:

Generation of monodisperse TPP-chitosan microparticles using a cross-flow microfluidic chip coupled with external crosslinking reaction. Control the size of TPP-chitosan emulsions by altering the relative sheath/sample flow rate ratio. The strategy is based on the sheath effect (focusing) to form uniform self-assembling sphere structures, so-called water-in-oil (w/o) emulsions, in the cross-junction microchannel. These fine emulsions, consisting of aqueous 1% (w/v) chitosan, are then dripped into a solution containing 10% (w/v) tripolyphosphate (TPP). Which is then undergo an ionic-crosslinking reaction and create water-insoluble TPP-chitosan microparticles in an efficient manner. The proposed microfluidic chip is capable of generating relatively uniform micro-droplets and has the advantages of actively controlling the droplet diameter and having a simple and low cost process with a high throughput¹⁴.

Cross-linking of chitosan microspheres with genipin on protein release:

Bovine albumin as a model protein was mixed with chitosan to make microspheres cross-linked by genipin. The effect of cross-linking time and genipin concentration on swelling ratio, the degree of cross-linking and the elution of albumin from the microspheres was evaluated.Chitosan microspheres have shown much potential in minimally invasive applications for local delivery of therapeutic agents and in composites for combined tissue engineering and drug delivery systems¹⁵.

Immobilization:

Chitosan is known as an ideal support material for enzyme immobilization because of its many characteristics like improved mechanical strength, resistance to chemical degradation, avoiding the disturbance of metal ions to enzyme, and anti-bacterial property. The properties of immobilized biocatalysts are influenced by the characteristics of enzyme, support material and the immobilization method. A suitable support should have high affinity to proteins, reactive functional groups, hydrophilicity, mechanical stability and rigidity. Chitosan is a natural polyaminosaccharide which offers the all characteristics therefore it is often used for enzyme immobilization.

Enzyme immobilization:

Enzyme immobilization technology is an effective means to perform enzyme reuse and to improve its stability. Using of soluble enzymes has several disadvantages, e.g. instability and sensitivity to process conditions other than the optimal ones. Therefore the application of solid-phase biocatalysts has become more and more important during the last decades. Advantages of immobilized enzymes are that the expensive biocatalysts can be used repeatedly in successive batches, or the process can eventually be carried out in a continuously operating reactor.

Enzymes can be immobilized on various supports and by different methods. For practical purposes, carrier beads with size falling into millimetre range are mainly used. However, more and more results are reported on immobilization of enzymes onto microparticles possessing high specific surface area and numerous active sites available for the enzyme molecules to be fixed. Chitosan a natural polyaminosaccharide obtained by N-deacetylation of chitin was selected as support. Different-sized chitosan support particles can be formed by several methods, e.g. precipitation, emulsion cross-linking, spray drying, ionotropic gelation, emulsion-droplet coalescence and reverse micellar method. Its reactive amino and hydroxyl groups after chemical modifications make possible the coupling of enzymes. Therefore, chitosan is often used as support material for enzyme immobilization¹⁶.

Immobilization of water-soluble metallophthalocyanine complexes:

The immobilisation of water-soluble metallophthalocyanine complexes on chitosan aerogel microspheres affords new bifunctional catalysts which have been used for the aerobic oxidation of β-isophorone. Chitosan gives the support of the metal complex and the organic base necessary for the reaction.

The immobilisation of metallophthalocyanines onto chitosan enables one to get a material having two kinds of sites. This material is a promising heterogeneous catalyst for the oxidation of b-isophorone to ketoisophorone. This novel method are having following advantages

1. Use dioxygen as an oxidant.

2. Use support from inexpensive renewable.

3. Combination of basic and oxidation sites in one solid material to avoid an addition of external base or other additives resulting in no wastes.

4. Easy separation of catalyst from reaction mixture and possibility of recycling [17].

Beta-galactosidase immobilization on chitosan microspheres:

Chitosan microspheres suitable for enzyme immobilization were prepared by emulsion cross-linking method. Aqueous chitosan solution was added dropwise into an oil phase consisted of sunflower oil and n-hexadecane. Cross-linking was performed with glutaraldehyde which makes possible the covalent attachment of enzymes. β-Galactosidase which is applied in dairy industry for lactose hydrolysis was immobilized onto the microspheres¹⁸.

For immobilization of dye:

Chitosan microsphere (CS) was prepared by phase-inversion method as the support matrices. Cibacron Blue F3GA (CB) was covalently attached to the chitosan microspheres, and thus the novel dye-affinity adsorbent was obtained. These Cibacron Blue F3GA-attached chitosan microspheres (CB-CS) were used in the catalase (CAT) adsorption studies¹⁹.

Biosensor based on laccase immobilized on microspheres of chitosan:

Laccase is a type of copper-containing oxidase which is widely distributed in fungi, higher plants and in some bacteria. It can oxidize polyphenols, anilines and benzenethiols with the concomitant reduction of molecular oxygen to water. This enzyme has been applied to many industrial processes including degradation of xenobiotics, effluent treatment, pulp delignification,decolorization of dyes, biopulping, biobleaching, synthetic chemistry, soil bioremediation, food processing, denim bleaching, cosmetics and biosensors . Laccase-based biosensors have been employed for the determination of a broad range of phenolic compounds. The construction of a biosensor based on laccase immobilized on microspheres of chitosan crosslinked with tripolyphosphate and used for rutin determination in pharmaceutical formulations²⁰.

Composite magnetic microspheres:

In various kinds of biochemical and biotechnological applications (such as immobilization of biomolecules, wastewater treatment, affinity chromatography, drug delivery systems) the separation and recovery steps are difficult and expensive with respect to conventional techniques. Magnetic carrier technology is a very promising alternative to enhance the operational performance of these steps. The most important parameters in magnetic carrier technology are the economy and physicochemical characteristics of the carriers.

Composite magnetic microspheres have been synthesized based on artemisia seed gum and chitosan using the suspension cross-linking technique for use in the application of magnetic carrier technology. Composite magnetic microspheres were prepared and characterized for application in magnetic carrier technology particular in enzyme immobilization and bioaffinity chromatography^{34, 35}.

Immobilization of laccase on magnetic chitosan microspheres:

Laccases have been successfully immobilized on many different types of carriers such as controlled porosity glass, activated carbon, oxirane acrylic beads, and hydrophilic PVDF microfitration membrane. The studies of laccase immobilization on magnetic chitosan microspheres have rarely been reported. Immobilization can be carried out by using glutaraldehyde to form the Schiff’s base. In recent years, magnetic carrier technology has showed significant attractive for the preparation of immobilized enzymes. Chitosan can be used as a base material for magnetic carriers. Used as the support material, magnetic carriers can be quickly separated from the reaction medium and effectively controlled by applying a magnetic field, thus the catalytic efficiency and stability properties of enzyme can be greatly improved³⁶.

Hemoglobin immobilization:

Magnetic chitosan microspheres (MCMS) have been prepared and applied in immobilization of tyrosinase and laccase. Carbon-coated iron nanoparticle is a kind of new magnetic nanomaterials which was obtained by coating a layer of carbon uniformly on the surface of nanoscale iron. So it can be resistant against corrosion and oxidation and has a small average size which makes it highly promising for applications. Most previous research on magnetic microspheres has focused on iron oxide as the magnetic core currently. However, few studies were reported on preparing magnetic microspheres using the magnetic core of carbon-coated iron nanoparticles and immobilization of peroxidases on them.

A novel magnetic chitosan microsphere (MCMS) was prepared using carbon-coated iron magnetic nanoparticles and chitosan. Hemoglobin (Hb) was successfully immobilized on the surface of MCMS modified glassy carbon electrode (GCE) with the cross-linking of glutaraldehyde and the immobilized Hemoglobin displayed an excellent electrocatalytic property to the reduction of H₂O₂ in the presence of the mediator of methylene blue (MB).An amperometric hydrogen peroxide biosensor was fabricated based on the immobilized Hemoglobin modified electrode with the mediator of methylene blue (MB)³⁷.

Chitosan microspheres for antioxidant delivery:

Olive-leaf extracts (OLE):

Polyphenolic compounds were found to exhibit highly potent anti-oxidant activity. Incorporation of polyphenolic compounds in various foods as nutraceuticals is a growing area of research. There has been no study on the encapsulation of polyphenolic compounds from olive-leaf extract (OLE) into chitosan microspheres. Encapsulation of OLE in the chitosan microspheres was achieved successfully by a spray-drying process. The activity of the polyphenolic compounds encapsulated through OLE in the chitosan matrix was found to be retained even after the spray-drying process²¹.

α-Lipoic acid:

Oxidative stress underlies both minor and severe cell damage with a range of physiological (e.g. muscle fatigue and tiredness) and patho-physiological consequences such as cardiovascular disease and cancer. α-Lipoic acid is an important and powerful biological antioxidant that can directly scavenge free radicals and protect cells from oxidative damage. Free α-lipoic acid is rapidly taken up by cells and reduced to dihydrolipoic acid (DHLA) intracellularly.

Supplemental doses of α-Lipoic acid (LA) are rapidly metabolized and rapidly cleared from plasma and tissues, suggesting that it should be taken in divided doses through out the day rather than a single dose. A constant plasma level of LA might be highly beneficial for various reasons such as given below:

(a) Immediate release spikes probably leading to toxic side effects can be avoided.

(b) The antioxidative and radical scavenging effect might be guaranteed for a prolonged period of time and not just a few hours.

Encapsulation of α-Lipoic acid using chitosan which was chosen as the encapsulant matrix due to its excellent biocompatibility and biodegradability. Lipoic acid encapsulated chitosan microspheres were prepared by spray-drying. The spray-dried chitosan microspheres obtained were of good morphological characteristics and a narrow size distribution. The encapsulation process may cause some structural interactions between lipoic acid and chitosan. Studies on antioxidant activity of encapsulated LA demonstrated a significant level of retention of activity when compared to free lipoic acid. The retention of the non-extractable LA in the chitosan matrix may provide a sustained release of the antioxidant for an extended period of time²².

Superoxide dismutase loaded chitosan

microspheres:

Superoxide dismutase (SOD) is the most potent antioxidant enzyme. SOD was encapsulated in chitosan microspheres to obtain suitable sustained protein delivery. Due to the inherent characteristic of SOD, high encapsulation efficiency could not be obtained with simple preparation method. The pH of chitosan solution is 3.0, when the chitosan microspheres were prepared with this solution, encapsulation was low. Therefore several strategies have been tested to increase the encapsulation efficiency and good results have been obtained²³.

pH sensitive microspheres:

pH-sensitive and mucoadhesive thiolated Eudragit-coated chitosan microspheres:

The Eudragit–cysteine conjugate and coating with chitosan for developing an oral protein drug delivery system, having mucoadhesive and pH-sensitive property. Bovine serum albumin (BSA) as a protein model drug was loaded in thiolated Eudragit-coated Chitosan microsphere to study the release character of the delivery system. After thiolated Eudragit coating it was found that the release rate of BSA from BSA-loaded TECMs was suppressed at pH 2.0 PBS solutions, while at pH 7.4 PBS solution the BSA can be sustainingly released for several hours. The structural integrity of BSA released from BSA-loaded TECMs was guaranteed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy.

BSA-loaded TECMs were prepared by coating negative charged thiolated Eudragit with BSA-loaded positive charged CMs. This system offers amild formulation procedure which benefits for delivery and therapeutic activity of polypeptides. The release of BSA from BSA-loaded TECMs were decreased compared with that of CMs and the higher amount of thiolated Eudragit coating further retarded the BSA release from micropsheres at pH 2.0. The mucoadhesive property of TECMs was significant higher than that of ECMs and CMs both in vitro and in vivo. Therefore, the TECMs will be suitable for oral protein drug delivery²⁴.

Nasal delivery/ pulmonary administration:

Conventionally the nasal route has been used for the delivery of drugs in the treatment of local diseases; however the last decade has recognised the importance of the nasal cavity as potential route for drug delivery, particularly of small molecular weight polar drugs, peptides and proteins. There is an increasing number of research and review articles addressing topics on nasal drug delivery. This interest arises from the different possible advantages presented by the nasal cavity, such as: the epithelium very vascularized and with a relatively large surface area available for drug absorption, the porous endothelial basement membrane, the direct transport of absorbed drugs into the systemic circulation thereby avoiding the first-pass effect hepatic present in peroral administration, the lower enzymatic activity compared with the gastrointestinal tract and the liver. For all these reasons the nasal route can be considered a useful alternative both to parenteral and oral routes. A wide range of nasal products is in development, mostly in correlation with the rapid onset of action of nasal route, for example, for the treatment of pain (nasal morphine and ketamine) and for the treatment of erectile dysfunction (nasal apomorphine).

A limitation of nasal drug delivery is the mucociliary clearance that determines a limited time available for adsorption within the nasal cavity. One strategy for increasing drug absorption is to prevent the rapid clearance of the delivery system from the nasal mucosa and thereby prolong the contact between the nasal mucosa and the formulation.Mucoadhesive materials can increase the time available for drug absorption. Chitosan is one of the materials that have been shown to be mucoadhesive. The free amino groups resultant from the deacetylation process of chitin enable the formation of positively charged chitosan salts with organic and inorganic acids. Chitosan may be a good option in nasal delivery as it binds to the nasal mucosal membrane with an increased retention time and it is a good absorption enhancer

Pulmonary administration of drugs to treat localized disease states within the bronchi has been employed for years. In this way the drugs can be delivered into the diseased regions, thus reducing side effects due to drug distribution to other organs. Inhalation of aerosolized drugs can also represent an ideal method for drug delivery to the systemic circulation. However, drug disposition in the lung following inhalation still depends on simultaneous processes, these are: deposition, absorption, metabolism and mucociliary clearance, and generally there is the need to frequently administer therapeutic dose (3–4 times daily), thus limiting patient compliance. Formulation strategies to prolong the drug’s retention time in the lung can be useful to improve or retard absorption, minimizing the biodistribution throughout the systemic circulation, thus influencing therapeutic effects and reducing side effects.Chitosan microparticles can be used for this purpose.

Chitosan has been shown to be degraded mainly by lysozyme, which commonly exists in various human body fluids and tissues and represents one of the most abundant enzymes in the lung, in which it is synthesized and secreted by glandular serous cells, surface epithelial cells, and macrophages in the human airways. Chitosan is able to form microparticulate carriers with good bioadhesive properties related to attractive electrostatic forces between the negative charged glycoprotein of mucin and the positive charged amino groups of the polymer. Moreover, by means of a reduction of mucociliary clearance and the opening of tight intercellular junctions, chitosan is able to increase drug bioavailability after nasal administration.

Chitosan microparticulate system for pulmonary administration of moxifloxacin:

Moxifloxacin (MXF), an 8-methoxy-quinolone with a broad spectrum of antimicrobial activity against common respiratory pathogens. The most common adverse events associated with MXF administration are gastrointestinal disturbances, such as nausea and diarrhea, due to the impact on the human intestinal microflora, mainly on the enterobacteria. These side effects can be avoided by loading the drug into a biodegradable microparticulate system suitable for inhalation. A bioadesive polymer used as carrier, such as chitosan, can reduce the number of MXF administrations, increasing patient compliance.

MXF-loaded chitosan microspheres were prepared by the spray-drying technique using different amounts of glutaraldehyde as crosslinking agent²⁵.

Mucosal immune responses of Bordetella

bronchiseptica DNT using chitosan microspheres:

In vitro immune-stimulating activities of Bordetella bronchiseptica dermonecrotoxin (BBD)-loaded in chitosan microspheres (CMs) were reported with a mouse alveolar macrophage cell line. Based on the report, in vivo activity of immune-induction was investigated by intranasal administration of the BBD-loaded CMs into mice. BBD was loaded into the CMs prepared by an ionic gelation process with tripolyphosphate. Mice were immunized by direct administration of the BBD-loaded CMs into the nasal cavity.After immunization of the mice; BBD-specific immune responses (IgG and IgA titers) were measured in sera, nasal wash, and saliva by ELISA. BBD-specific IgA titers in the nasal cavity were time- and dose-dependently increased by the administration. Similar phenomena were observed in the analysis of systemic IgA and IgG in sera. These results suggested that direct vaccination via the nasal cavity was effective for targeting nasal-associated lymphoid tissues, and that CMs were an efficient adjuvant in nasal mucosal immunity for atrophic rhinitis vaccine²⁶.

Intranasal delivery of Bordetella bronchiseptica antigens containing dermonecrotoxin:

Respiratory infection of the nasal cavity of pigs by B. bronchiseptica is a common risk factor leading to the disease, atrophic rhinitis (AR). The turbinate atrophy of pigs develops only after infection with a B. bronchiseptica strain producing dermonecrotoxin. Evaluated the potential use of Chitosan microspheres (CMs) in the presence of F127 as drug delivery system for intranasal delivery of the B. bronchiseptica multiple antigens containing dermonecrotoxin (BBD)²⁷.

Microencapsulated chitosan nanoparticles for lung protein delivery:

Prepared Chitosan/tripolyphosphate nanoparticles that promote peptide absorption across mucosal surfaces. To microencapsulate protein-loaded chitosan nanoparticles using typical aerosol excipients, such as mannitol and lactose, producing microspheres as carriers of protein-loaded nanoparticles to the lung. The results showed that the obtained microspheres are mostly spherical and possess appropriate aerodynamic properties for pulmonary delivery.

Protein-loaded nanoparticles can be successfully incorporated in microspheres by means of a spray-drying process, resulting in dry powders with suitable properties for lung delivery. The mannitol/nanoparticles ratio significantly affects the microspheres morphology, showing improved spherical shapes with increasing amounts of nanoparticles. Growing concentrations of the spray-drying suspensions led to an increase in the particles aerodynamic diameter. Recovering of nanoparticles from microspheres is efficiently conducted in vitro after incubation in an aqueous medium. Therefore, after contact with the lung aqueous environment, microspheres are expected to release the nanoparticles and, as a consequence, the therapeutic macromolecule. This system is proposed for systemic delivery of therapeutic macromolecules, considering the already known properties of chitosan to promote peptide absorption. In addition, it could also be used as a tool in therapy of lung local diseases, such as cystic fibrosis or cancer²⁸.

Pulmonary delivery of therapeutic proteins:

Recently developed a new drug delivery system consisting of complexes formed between preformed chitosan/tripolyphosphate nanoparticles and phospholipids, named as lipid/chitosan nanoparticles (L/CS-NP) complexes. These protein-loaded L/CS-NP complexes were prepared by spray-drying, using mannitol as excipient to produce microspheres with adequate properties for pulmonary delivery. Microspheres are spherical and present appropriate aerodynamic characteristics for lung delivery resulting with adequate properties to provide a deep inhalation pattern²⁹.

Nasal administration of Carbamazepine using chitosan microspheres:

Carbamazepine is a drug widely used as antiepileptic agent, in the therapy of psychomotor seizures and trigeminal neuralgia. It is traditionally given by oral administration but due to its poor water solubility (about 170 mg/l at 24⁰C) it is characterized by slow and irregular gastrointestinal absorption. Hepatic first-pass effect due to the enzymatic auto-induction of its metabolism. This latter characteristic besides the need of a therapeutic prompt action make CBZ a possible candidate for the development of a nasal formulation. The microspheres were prepared by a spray-drying method³⁰.

Chitosan-ethylcellulose microspheres for nasal delivery:

Loratadine-loaded microspheres were prepared by spray-drying of dispersions, emulsions and suspensions differing in polymeric composition and solvents used. Conventional microspheres were obtained by spray-drying of dispersions composed of chitosan (CM) as only polymer, while composed microspheres were obtained by spray-drying of two-phase systems composed of chitosan and ethylcellulose (EC). All microspheres were positively charged, indicating the presence of chitosan at the surface, regardless of the drug content and the type of spray-dried system. Tensile studies showed that both, EC/CM ratio and the type of spray-dried system influenced the bioadhesive properties of the microspheres in a way that the microspheres with higher chitosan content were more bioadhesive and microspheres prepared from suspensions were more bioadhesive than those prepared from emulsions, regardless of the same polymeric composition. Composed microspheres should ensure longer retention of the drug delivery system at the site of deposition, as loratadine was significantly less present at their surface, and consequently had less influence on bioadhesion. Higher chitosan content (EC/CM 1:3) ensured more compact coating of EC cores of microspheres, improving their bioadhesive properties³¹.

Magnetic microspheres:

Thiourea-modified magnetic chitosan microspheres:

Chitosan is capable of adsorbing a number of metal ions as its amino groups can serve as chelation sites. Due to their high nitrogen content and porosity, chitosan-based sorbents have exhibited relatively high sorption capacities and kinetics formost heavy metals. However, lack of specificity toward several highly toxic heavy metals limits the use of chitosan as an effective sorbent. The thiourea-modified magnetic chitosan microspheres (TMCS) were prepared. The high content of amino groups makes possible chemical modification in magnetic chitosan with the purpose of improving its features as an adsorbent, such as selectivity and adsorption capacity³².

Chitosan–poly (acrylic acid) polymer- CS–PAA magnetic microspheres:

Magnetically controlled drug targeting is one of the various possibilities of drug targeting. This technology is based on binding targeted drugs with magnetic nanoparticles, which concentrate drugs in the area of interest by means of magnetic fields. Recently, the development of magnetically responsive microspheres has brought an important driving force into play. Different inorganic or polymeric materials have been proposed as carriers of magnetic materials. A considerable advantage of the polymeric carriers is the presence of a variety of functional groups, which is able to modulate the carrier properties for the desired applications. Stable CS–PAA polymer magnetic microspheres with high Fe3O4 loading content were prepared. These nanoparticles were prepared by cationic CS coating negatively charged Fe3O4 nanopartilces by electrostatic adsorption and subsequent polymerization of acrylic acid (AA) onto the CS-coated Fe3O4 cores.

These polymer magnetic microspheres had a high Fe3O4 loading content, and showed unique pH-dependent behaviors on the size and zeta potential. A continuous release of the entrapped ammonium glycyrrhizinate in such polymer magnetic microspheres occurred, which confirmed the potential applications of these microspheres for the targeted delivery of drugs³³.

Application in tissue engineering approaches:

Tissue engineering has emerged as an important alternative approach to autografts and allografts. It has been defined as the application of biological, chemical and engineering principles towards the repair or regeneration of living tissues using biomaterials, cells and factors alone or in combination. Many tissue engineering methods involve using a biocompatible and biodegradable three-dimensional (3-D) polymeric scaffold as a temporary extracellular matrix for initial cell attachment and subsequent tissue formation. The scaffold should be porous, with interconnected pore networks for cell growth and transport of nutrients and metabolic wastes. Pore sizes and their distribution were identified as the first parameter that generates a capillary phenomenon, giving rise to osmotic pressure between the inner and outer sides of the implant. Macropores are expected to be more reactive while micropores in the range of 5 lm or less play an important role in controlling the rate of bioresorbability.

Several natural and synthetic polymers have been used as scaffolding materials for bone tissue engineering. Natural polymers include type I collagen, hyaluronic acid and chitosan. Synthetic polymers include poly (lactic acid) (PLA), poly (glycolic acid) (PGA) and their copolymer, poly (lactic acid–glycolic acid) (PLAGA). Natural biopolymer chitosan has been investigated for a variety of biomedical applications, such as wound healing and bone and cartilage tissue engineering, due to its excellent biocompatibility and biodegradability.

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. Development novel 3-D chitosan/ poly (lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.

These composite scaffolds possess excellent mechanical properties attributed to the PLAGA component. In addition, chitosan introduces functionality to the composite scaffolds due to its reactive groups and further bioactivity may be introduced to the composite scaffolds^{39, 40}.

Multimicrospheres (CCAM) formation:

Multimicrospheres (CCAM) with hydrophilic core and hydrophobic coating as drug delivery system .Cellulose acetate was selected as hydrophobic coating to entrap the hydrophilic chitosan microcores. During the preparation of the CCAM, no chemical crosslinking agent was used. Cellulose acetate, which made from a renewable resource of processed wood pulp was widely used in oral pharmaceutical products and was regarded as a nontoxic, nonirritant and biodegradable material. Cellulose acetate had been used as a semipermeable coating on tablets, especially on osmotic pump-type tablets and implants, which allows for controlled, extended release of actives.

Ranitidine hydrochloride (RT), acetaminophen (ACP) and 6-mercaptopurine (6-MP) were selected as hydrophilic, amphoteric and hydrophobic model drugs respectively. RT was a competitive, reversible inhibitor of the action of histamine on the histamine H2-receptors. ACP was a popular analgesic and antipyretic drug that was used for the relief of fever, headaches, and other minor aches and pains. 6-MP was a purine analogue, which was antimetabolite and antineoplastic agent. Chitosan, cellulose acetate and model drugs with different hydrophilicity could be made into microspheres by the methods of W/O/W emulsification and solvent evaporation. The CCAM were spherical, free-flowing and non-aggregated. With the increasing of hydrophobicity of drug, the holes in the appearance of CCAM became smaller and almost disappeared. The CCAM system had good effect on the controlled release of different model drugs with different hydrophilicity⁴¹.

In targeted delivery:

Targeted delivery of therapeutic agents has been well recognized for its potential advantages in enhancing drug therapeutic efficacy and reducing side effects. There are growing interests in developing delivery systems for drug targeting to liver cells because of the lack of other effective and practical pharmacological approaches.

Particle size is critical for passive targeting, whereas active targeting systems require the need for the receptor to trap specific ligands. Liposomes and microspheres are two examples of passive delivery systems for liver chemotherapeutic agents. Asialoglycoprotein receptors (ASGR) including the galactose receptor are a group of well known surface receptors present only in hepatocytes and several human hepatoma cell lines. A ligand containing galactose moiety can be recognized by and bound to the liver-specific galactose receptor. The ligandreceptor complex is uptaken rapidly into the cells, and the receptor recycles back to the cellular surface. In other words, the galactose receptor has a high binding capacity and leads to efficient cellular uptake of galactosylated ligands. The galactose receptor mediated endocytosis makes this receptor an ideal target for developing active targeting delivery systems for hepatocyte /liver and hepatoma cells.

Because of good film forming properties, chitosan also was used to coat the microsphere composed of poly (lactic acid)–poly (caprolactone) blends.

Few of lactosaminated or galactosylated chitosan derivatives were chemically conjugated with drugs. For example, lactosaminated N-succinyl-chitosan was utilized in mice as drug-specific to the liver. Galactosylated chitosan-graft-dextran, galactosylated chitosan–graft–poly (ethylene glycol) and galactosylated chitosan-graft-PVP had excellent specificity to liver cells as hepatocyte-targeting DNA carrier. The galactosylated chitosan microspheres were prepared and demonstrated for their utilization for active targeted drug delivery to liver.

Bovine serum albumin (BSA) is a natural biocompatibility, nontoxic, nonantigenic microspheres forming material. We expect that the BSA microspheres with negative charges may strongly adsorb soluble galactosyl chitosan with positive charges so that the liver targeting properties on its surface may be significantly changed. 5-Fluorouracil (5-FU), one of the primary drugs used for the liver cancer treatment, is selected as the model drug. The 5-FU loaded BSA micropheres with galactosyl chitosan coating may possess both passive and active targeting properties against liver cells, and potentially improve the safety and efficiency⁴².

Microspheres to target macrophage mannose receptors:

The mannose receptors are predominantly present on macrophages and dendritic cells, which play a central role in innate and adaptive immune responses. Mannose is highly expressed on the surface of many pathogenic micro-organisms, such as bacteria and yeast, and the mannose receptors are involved in receptor-mediated endocytosis and phagocytosis. Macrophage mannose receptors (MMR; CD206) bind oligosaccharides containing terminal mannose residues on infectious agents and transport them into endocytic pathways, which results in MHC presentation and subsequent T cell activation. Therefore, the mannosylation of drugs or a delivery system may correlate with improvement of receptor-mediated uptake upon binding MMR. Currently, mannosylation of drugs has been reported to enhance MHC class I- and II-restricted antigen presentation and T cell stimulation compared with non-mannosylated proteins. Furthermore, mannosylated-drug delivery systems such as mannosylated chitosan/ or chitosan nanoparticle, liposome and niosome have been demonstrated to augment immunogenicity by targeting mannose receptors on antigen-presenting cells. Therefore, mannosylation of drugs or their delivery systems can be exploited as a promising strategy to enhance immunogenicity because of their ability to target MMR.

Mucosal delivery is a well-documented and highly effective route for the stimulation of local and systemic immunity. However, soluble drugs are usually poor immunogens when administered by mucosal routes and require the adjunct of a mucosal adjuvant or a drug delivery system. Recently, chitosan has received attention as an adjuvant for mucosal delivery by facilitating higher drug and carrier bioavailability. Chitosan is used in multiple biomedical and pharmaceutical applications due to factors such as biocompatibility, biodegradability, high charge density, and non-toxicity. Chitosan microspheres (CMs) are the most widely studied drug delivery system for the controlled release of various drugs. Nano or microparticles of chitosan are easily formed and are under investigation for the mucosal delivery of antibiotics, antihypertensive agents, proteins, peptide drugs, and vaccines. CMs have previously been shown to enhance the mucosal absorption of various compounds, such as drug delivery system, and have adjuvant activity in the mucosal immune response by intranasal administration ⁴³.

Application of hollow chotosan microspheres:

Recently, hollow microspheres have attracted great attention because of a variety of applications such as delivery vesicles for drugs, DNA, antigens, and protection proteins and enzymes, especially for controlled or sustained drug-delivering systems employing biopolymers as raw material

The poor solubility of chitosan at neutral pH and higher limits its application as an adsorption enhancer in the basic environment of the large intestine, colon, and rectal mucosa. Moreover, some model drugs would be damaged by the acidic solution system of dissolved chitosan, the chitosan only can be dissolved in acidic conditions for the formation of the microspheres. To overcome these disadvantages of classical chitosan solutions, it is essential to prepare chitosan derivatives which are water-soluble around neutral pH. Some progresses have been reported in the preparation of water-soluble chitosan derivatives, such as: N-lauryl-Nmethylene phosphonic chitosan, a-D-mannoside branches at C-6 of chitin and chitosan, tosyl- and iodo-chitin, N-carboxymethylated chitosan, N-acetylchitosan, and N-trimethyl chitosan chloride.

The preparation of hollow spheres of chitosan using cyclohexane droplets as template, and NMC cross-linked with glutaraldehyde as the shell. An oil-in-water emulsion system was chosen to prepare the hollow microspheres with smooth surfaces, because of the wide variability in both composition and physical parameters of water and oil phases and its potential for industrialization. The unique structure and function of microspheres was attractive for their potential for encapencapsulation of large quantities of guest molecules or large size guest molecules within the ‘‘empty’’ core domain. A series of hollow microspheres encapsulated with ofloxacin have been successfully prepared, using cyclohexane droplets as a template and the N-methylated chitosan (NMC) cross-linked with glutaraldehyde as the shell. A new multiparticulate system, with the potential for site-specific delivery to the colon, has been developed using ketoprofen as model drug. The system simultaneously exploits cyclodextrin complexation, to improve drug solubility⁴⁴.

Enhancement of immunogenicity by Chitosan microspheres:

Tuberculosis (TB) remains a major health problem worldwide. One-third of the world population is infected with Mycobacterium tuberculosis (Mtb), and approximately 5–10% of those infected become sick or infectious at some time during their lifetime. Moreover, the percentage in which progressive disease develops has been increasing markedly in recent decades due to the spread of HIV/AIDS and the emergence of multidrug-resistant TB.

Over the last decade, biocompatible and biodegradable material, chitosan and its nano- and microparticles were investigated for delivery of hydrophilic macromolecules such as peptide protein, drugs and vaccines. Chitosan and its microspheres have many advantages for vaccine delivery. First, chitosan could open the intercellular tight junctions and favour the paracellular transport of macromolecules. Second, chitosan nano- and microparticles are suitable for controlled drug and vaccine release. Third, chitosan nano- and microparticles are most efficiently taken up by phagocytotic cells. Thus chitosan and its derivatives could induce strong systemic and mucosal immune responses against antigens. Nasal chitosan influenza vaccine was both effective and protective in human and nasal chitosan pertussis, diphtheria, and atrophic rhinitis vaccines also have good efficacy in animal models.

Besides enhancing the immune response by opening the intercellular junctions or stimulating the uptake by macrophages, chitosan may also stimulate the immune system as adjuvant. Partially deacetylated chitin was effective in activation of macrophages after intraperitoneal administration in mice; and 70% deacetylated chitin was shown to possess adjuvant activity for induction of cell-mediated and humoral immunity. Furthermore, chitosan stimulated the induction of cytokines like interleukin, interferon, and colony stimulating factor after intraperitoneal injection in mice. Only phagocytosable chitosan particles were able to induce significant interferon gamma levels and alveolar macrophage priming after intravenous administration. These studies indicate that chitosan particles could stimulate macrophages, B and T lymphocytes. Therefore, chitosan nano- and microparticles used as immunological adjuvants or vaccine carriers are promising to induce both humoral and cellmediated immunity⁴⁵.

Antibacterial activity of chitosan microspheres:

Chitosan is known for its antibacterial properties, higher killing rate, and lower toxicity toward mammalian cells, not only possess a wide inhibition spectrum against Gram-positive and Gram-negative bacteria, but also sterilize some yeasts and moulds.

Since chitosan is insoluble inmost solvents but is soluble insome dilute organic acids, most of researches on the antibacterial activities of chitosan and its derivatives against microbe were focused on the invisible solution molecule interaction. Because of the positive charge on the C-2 of the glucosamine monomer below pH 6, interaction between positively charged chitosan molecules and negatively charged microbial cell membranes leads to the leakage of pertinacious and other intracellular constituents. Dissociated chitosan molecule in solution, with lower molecular weight , could bind with DNA and inhibit synthesis of mRNA through penetration toward the nuclei of the microorganisms and interfere with the synthesis of mRNA and proteins .While with higher molecular weight, the dissociated chitosan molecule could interact with the membrane of the cell to alter cell permeability , or acts as a chelating agent that selectively binds trace metals and thereby inhibits the production of toxins and microbial growth . Compared with the dissociated chitosan molecule could interact with microbe sufficiently in solution, chitosan solid matrix, such as beads, films, fibers, and hydrogels. Chitosan contact bacterial outer membrane (OM) firstly, after series of physical and biochemical interactions, kill the bacterial finally. However, few authors had applied the chitosan microspheres (CMs) to the antibacterial studies.

In the field of antimicrobial agent, fatty acids act as the key ingredients of antimicrobial food additives due to their inhibitory action on undesirable microorganisms, and the long-chain unsaturated fatty acids such as linoleic and oleic acids are bactericidal to important pathogenic microorganisms. Moreover, prolongation of the hydrocarbon chain was proven to increase the antibacterial activities against the tested bacterium. Based on the reports, hydrophobic modifications of the surface probably endow the CMs with superior antibacterial activities⁴⁷.

Adsorption of metal ions from environment:

Physical, chemical and biological approaches have been employed in the treatment of effluents containing high contents of metals and organic matter. Adsorption is a promising alternative to remove metals from aqueous environments, especially when natural biodegradable adsorbents produced from biomasses are used. Various studies have aimed at selecting natural adsorbent materials which combine low-cost and high efficiency to remove metals from aqueous media.

Biopolymers isolated from marine organisms are promising adsorbents to remove heavy-metal ions from aqueous waste streams. Chitosan derived from chitin, is a major component of crustacean shells and the second most abundant natural biopolymer after cellulose. It contains a high percentage of reactive amine groups which assist its modification and make it highly selective when modified with chelating agents.

The introduction of selected functional groups into the polymeric matrix of chitosan enhances its interaction with a variety of metallic ions, improving its selectivity and specificity, therefore increasing its adsorption capacity. Chemical modifications by cross-linking agents increase the chemical stability of the sorbent in acid media and especially decrease its solubility in most mineral and organic acids. Cross-linking may be performed by the reaction of chitosan with different crosslinking bi-functional agents such as glutaraldehyde, ethylene glycol diglycidyl ether, glycerolpolyglycidylether or hexamethylenediisocyanate. It is also possible to use mono-functional reagents, such as epichlorohydrin or chloromethyloxirane. Tri-polyphosphate has also been selected as a possible cross-linking agent, which can be used in the preparation of chitosan gel beads.

Use of heparin to modify the surface of chitosan as a result of the ionic interaction between these two biopolymers. The addition of functional carboxylate and sulfonate groups, in addition to amine and hydroxyl groups may enhance the interaction of the material with copper (II) ions in aqueous solutions thus increasing the adsorption capacity of such novel bioadsorbent.The material was cross-linked with epichlorohydrin to increase its resistance to acid solutions⁴⁸.

Extraction of metal ions:

The extraction, beneficiation and use of mineral coal produce effluents of elevated acidity (pH ≈3.0) and significant concentration of metals, such as Fe, Al, Mn, Cu, Zn and Pb. These effluents are hazardous to the environment and may contaminate hydro resources, thus threatening the integrity of living organisms.

Chitosan is biopolymer is capable of both neutralizing and removing iron, aluminum and copper ions from such effluents. Evaluating the use of chitosan microspheres for their importance in continuous systems. The microspheres were prepared by the phase inversion method. Their average diameter and morphology were determined. Water samples from decantation pool (DP) and acidic mine drainage (AMD) effluents were treated using different amounts of microspheres. The pH and concentration of Fe, Al and Cu ions were evaluated both before and after treatment of effluent samples

The chitosan microspheres are an alternative for the removal of acidity, Fe (III), Al (III) and Cu (II) from coal mining wastewater³⁸.

Chitosan microspheres for colonic delivery system:

In the treatment of inflammatory bowel disease (IBD), 5-aminosalicylic acid (5-ASA) and steroidal or non-steroidal anti-inflammatory drugs are frequently administered orally to the patients. Administration of these drugs at a large and frequent dose for a long period causes significant and prolonged absorption of the drugs from the small intestine, often leading to toxic side effects. Therefore the specific delivery of drugs to diseased parts has been developed. For example salazosulfapyridine, a prodrug of 5-ASA and Pentasa, acting as a delayed release system of 5-ASA, are clinically available. However they are not necessarily satisfactory and more improved systems are expected.

Recently a chitosan capsule containing 5-ASA was demonstrated to display an excellent effect against 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. Also micro- or nano-particulate dosage forms have been found to be effective to deliver drugs to the intestine, Peyer’s patches or colon. Small particles can penetrate the mucus layer more deeply and reach the diseased sites well. In these systems biocompatible and biodegradable polymers are often used and their degradation by enzymes of bacteria and macrophages in the diseased region can accelerate drug release.

In order to produce microparticulate system with the ability to show the gradual drug release and efficient delivery, synthesize a chitosan-succinyl-prednisolone conjugate (Ch-SP) as a macromolecular prodrug of prednisolone, and prepared the microspheres (Ch-SP-MS) using Ch-SP. In this study, novel preparation conditions were examined to obtain refined Ch-SP-MS and their Eudragit L100- or S100-coated Ch-SP-MS. Both types of Eudragit-coated Ch-SP-MS, protected Ch-SP-MS from morphological change at pH 1.2, and regenerated Ch-SP-MS fast at pH 6.8 and 7.4. For all types of microparticles, release of prednisolone was suppressed at pH 1.2, but caused gradually at pH 6.8⁴⁹.

Use of chitosan microspheres for cold injury:

Brain edema is still a critical problem that arises as a result of ischemia, infection, brain tumors, and traumatic injury. This situation is described as a pathophysiological condition of water content increase leading to the elevation of intracranial pressure. Mechanical failure of the tight endothelial junctions of the BBB causes transvascular leakage, resulting in vasogenic edema.

Vasogenic brain edema is the most commonly experienced type. An experimental model for the simulation of vasogenic type has been demonstrated by numerous investigators. The cold injury model has been used to demonstrate the vasogenic type brain edema. Membrane phospholipid degradation, lipid peroxidation, and breakdown of BBB are involved in vasogenic edema as the major pathophysiological situations.

Recently the effect of steroids in treatment of brain edema has become a focus of attention. The pathway of the steroids in treatment of brain edema is mainly dependent on the restoration of BBB, membrane stabilization, and degradation of the cell membrane phospholipids. The prevention of the intracellular lysosomal lytic enzyme activation, production of free radicals, which can easily react with the cell membrane, flux of the ions into the intracellular space, and formation of prostaglandins are also possible mechanisms of action in steroid treatment of brain edema. The effect of steroids on the secondary edema associated with the brain tumor is through reduction of the migration of proteins in tumoral and peritumoral tissues.

Microspheres is one of the novel drug carrier dosage forms, have recently become advantageous for drug delivery. Briefly, microspheres are defined as the microparticular dosage forms that can deliver the active ingredient to the targeted and/or desired site of action in the body. The chitosan microspheres were prepared using solvent evaporation method .The resulting microspheres provide a release for a period dependent upon their own physicochemical properties and those of the active substance. The dose of the active ingredient incorporated into microsphere formulations is lower than the systemic dose because the drug can be targeted directly to the site of action. There is no first-pass effect of degradation for the active ingredient. The side effects of the active material are minimized because of encapsulation into the microsphere formulations⁵⁰.

In embolotherapy:

Embolization, the process in which a blood vessel or organ is obstructed by the lodgement of a material mass, has been used in cancer treatment. Introduction of embolic materials into the blood vessels leading to a tumor diminishes its blood supply, thus starving the tumor. Spherical chitosan particles having an average diameter of 50–1000 mm were used for embolization of blood vessels by inserting a catheter into the blood vessel and injecting the particles through the catheter. After injection it was difficult to locate the injected embolic material and to monitor changes in its structure over time. Therefore Super paramagnetic iron oxide (SPIO) nanoparticles were developed for clinical applications in magnetic resonance imaging (MRI) contrast enhancement. The SPIO nanoparticles have the advantage of producing an enhanced proton relaxation in MRI, especially useful for T2-weighted images. We used a sonochemical method to synthesize SPIOnanoparticles with narrow size distribution and high magnetization⁵¹.

Chitosan microspheres for controlled release of different drug substances:

Chitosan microspheres (CMs) most widely studied drug delivery system for the controlled release of drugs such as antibiotics, antihypertensive agents, proteins, peptide drugs, and vaccines.

Antibiotics-loaded chitosan microspheres:

Emergence of newer antibiotics to combat bacterial resistance has been effective in controlling pathogens. The broad spectrum of activity against various pathogens involves structural interaction with bacterial receptors or blocking the metabolic function of microbes.

Doxycycline:

Doxycycline is a semi-synthetic antibiotic from Streptomyces species and possesses vicinal diols capable of binding with divalent ions and arresting the metabolic function. Doxycycline elicits its antimicrobial activity by preventing the addition of amino acids to growing peptide chains in bacteria. Apart from antimicrobial properties doxycycline also acts as an inhibitor of matrix metalloproteinases (MMPs), which is attributed to its metal-binding property. Evidence from animal studies shows that treatment with doxycycline improves healing parameters, like increasing the tensile strength of rat intestinal anastomoses after surgery and reducing the incidence of ulceration in alkali-injured rabbit eyes.

Doxycycline therapy at sub-antimicrobial dose has been shown to reduce periodontal disease activity by reducing MMPs and pro-inflammatory cytokines. Doxycycline inhibits host-derived MMPs by mechanisms independent of their antimicrobial properties. It is safe and effective at the dosages typically used in clinical practice and successfully tested in several conditions associated with elevated MMP activity. Doxycycline has been shown to reduce MMP activity in arthritis, periodontitis and aortic aneurysms. Adjunctive sub-antimicrobial dose doxycycline therapy can also improve the clinical parameters by controlling the level of MMP 8 in chronic adult periodontitis.

Chitosan microspheres were prepared by ionic gelation with KOH as crosslinking agent. The spheres were prepared by a w/o emulsion technique and are further utilized to encapsulate doxycycline.Doxycycline can elicit both antimicrobial and an MMP inhibition activity which is advantageous in wound healing application. Since chronic infected wounds, require a therapy to arrest infection and matrix degradation in a controlled fashion, the chitosan microspheres-loaded doxycycline would be an effective tool to develop therapeutic dressing encompassing both antimicrobial and MMP modulating ability⁴⁶.

Acyclovir:

Semi-interpenetrating polymer network (IPN) microspheres of acrylamide grafted on dextran (AAm-g-Dex) and chitosan (CS) were prepared by emulsion-crosslinking method using glutaraldehyde (GA) as a crosslinker.Acyclovir, an antiviral drug with limited water solubility, was successfully encapsulated into IPN microspheres by varying the ratio of AAmg-Dex and CS, % drug loading and amount of GA. In vitro release studies indicated the dependence of drug release rates on both the extent of crosslinking and amount of AAm-g-Dex used in preparing microspheres; the slow release was extended up to 12 hr⁵².

Ampicillin:

The solubility of non cross-linked chitosan in weak acid solutions restricts its utility in microspheres for drug delivery. To produce pentasodium tripolyphosphate cross-linked chitosan microspheres with higher acid resistance for controlled release of ampicillin. The microspheres were prepared by two different microencapsulation procedures (by emulsification and by spray-drying).Stability of uncross-linked and cross-linked microspheres was affected by the pH of simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.5). The inclusion of the enzymes pepsin and pancreatin did not affect the stability of the microspheres. The inclusion of lysozyme in phosphate buffer saline resulted in increased solubilization. The release of the drug was affected by cross-linking of microspheres with tripolyphosphate (TPP). The cross-linked microspheres were more stable in simulated gastric fluid and showed slower but sustained release of ampicillin⁵³.

Capecitabine:

The synthesis of capecitabine-loaded semi-interpenetrating network hydrogel microspheres of chitosan-poly (ethylene oxideg-acrylamide) by emulsion crosslinking using glutaraldehyde. Poly (ethylene oxide) was grafted with polyacrylamide by free radical polymerization using ceric ammonium nitrate as a redox initiator. Capecitabine, an anticancer drug, was successfully loaded into microspheres by changing experimental variables such as grafting ratio of the graft copolymer, ratio of the graft copolymer to chitosan, amount of crosslinking agent and percentage of drug loading in order to optimize process variables on drug encapsulation efficiency, release rates, size and morphology of the microspheres. In vitro release studies were performed in simulated gastric fluid (pH 1.2) for the initial 2 h, followed by simulated intestinal fluid (pH 7.4) until complete dissolution. The release of capecitabine was continued up to 10 h.⁵⁴.

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VB Yadav* and AV Yadav