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Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review

Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated... International Journal of Nanomedicine Dovepress open access to scientific and medical research Open Access Full Text Article R E v IEW Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review 1–3, Mohammad Ariful Islam * Abstract: Chitosan, a natural biodegradable polymer, is of great interest in biomedical research 1–3, due to its excellent properties including bioavailability, nontoxicity, high charge density, and Jannatul Firdous * mucoadhesivity, which creates immense potential for various pharmaceutical applications. It Yun-Jaie Choi 1–4 has gelling properties when it interacts with counterions such as sulfates or polyphosphates Cheol-Heui Yun and when it crosslinks with glutaraldehyde. This characteristic facilitates its usefulness in the 1,2 Chong-Su Cho coating or entrapment of biochemicals, drugs, antigenic molecules as a vaccine candidate, Department of Agricultural and microorganisms. Therefore, chitosan together with the advance of nanotechnology can Biotechnology, Research Institute for be effectively applied as a carrier system for vaccine delivery. In fact, chitosan microspheres Agriculture and Life Sciences, Center for Food and Bioconvergence, World have been studied as a promising carrier system for mucosal vaccination, especially via the Class University Biomodulation oral and nasal route to induce enhanced immune responses. Moreover, the thiolated form of Program, Seoul National University, Seoul, South Korea chitosan is of considerable interest due to its improved mucoadhesivity, permeability, stability, and controlled/extended release profile. This review describes the various methods used to *These authors contributed equally to this work design and synthesize chitosan microspheres and recent updates on their potential applications for oral and nasal delivery of vaccines. The potential use of thiolated chitosan microspheres as next-generation mucosal vaccine carriers is also discussed. Keywords: chitosan microspheres, oral, nasal, vaccine delivery, mucosal and systemic immune responses Introduction Vaccination is cost-effective, and probably the best preventable strategy against most diseases. Traditionally, vaccines are administered parenterally via an intramuscular 2,3 or subcutaneous route. This process of vaccine delivery incurs difficulties such as needle phobia, low patient compliance, short half-life, potential contamination while using needles, and a necessity for highly trained personnel. As a result, oral and nasal vaccination has been paid considerable attention as a way to overcome such potential drawbacks and eliminate the problems associated with parenteral administration of vac- Correspondence: Cheol-Heui Yun; cines. Better yet, parenteral vaccination mostly stimulates systemic immunity, whereas Chong-Su Cho mucosal vaccination tends to confer both systemic and mucosal immune responses. Department of Agricultural Biotechnology and Research Institute In regard to mucosal administration of protein drugs or vaccines, microspheres are for Agriculture and Life Sciences, 6–8 well known for their controlled delivery formulation, which would provide a long- Seoul National University, 1 Gwanak-ro, Gwanak-gu, lasting boosting effect and enhance the effectiveness of the immune response against Seoul 151-921, South Korea infectious diseases. Tel +82 2 880 4802 (CHY); +82 2 880 4868 (CSC) Chitosan has well-defined properties including bioavailability, biocompatibility, Fax +82 2 875 2494 (CSC) low cost, and an ability to open the intracellular tight junction; therefore, it has been Email [email protected] (CHY); [email protected] (CSC) suggested as a suitable polymeric material for mucosal delivery. Desirable properties submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 6077–6093 Dovepress © 2012 Islam et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article http://dx.doi.org/10.2147/IJN.S38330 which permits unrestricted noncommercial use, provided the original work is properly cited. Islam et al Dovepress of chitosan can be determined from its molecular weight CH (MW) and degree of deacetylation (DD). It has been reported O that high MW chitosan enhances the absorption of various NH CH OH 9,10 compounds across the mucosal barrier. Due to its cationic O O OH property, positively charged chitosan would have an elec- OH trostatic interaction with the negatively charged mucosal surface. Moreover, chitosan possesses mucoadhesivity, CH OH NH beneficial for prolonging the retention time at the mucosal area for a controlled and sustained therapeutic effect. CH n Nontoxicity is another prerequisite property of chitosan, Chitin which can be effectively applied for mucosal delivery of vaccines as a form of the microparticulate system. In an CH OH CH OH CH OH aqueous environment, chitosan swells and forms a gel- 2 2 O O O like layer, favorable for the interaction of polymers with OH O O glycoprotein in mucous. In the case of nasal delivery, OH OH OH OH chitosan possesses good bioadhesive properties and can NH NH NH 2 2 n reduce the rapid clearance of vaccine from the nasal cavity 2 where it could be delivered to nasal-associated lymphoid tissue – the induction and effector sites for vaccine-induced Chitosan immune responses. Figure 1 Structure of chitin and chitosan. General aspects of chitin Chitosan microspheres (CMs) and chitosan Extensive research has been carried out to exploit the use of Chitin is an abundant source of chitosan, a unique cationic chitosan as a drug or vaccine carrier. Indeed, chitosan has p o ly s a c c h a r i d e s u p e r i o r t o a ny m a n - m a d e c a t i o n i c been used for prolonged and targeted delivery of drug and derivatives. In general, it comprises the skeletal materials macromolecules. CMs can be a better option due to their in invertebrates. It is also found in egg shells of nematodes ability for sustained release and improved bioavailability of and rotifer as well as in the cuticles of arthropods, exo- target molecules. CMs also enhance the uptake of hydrophilic skeletons, peritrophic membranes, and cocoons of insects. substances across epithelial cells. It has been reported In the fungal walls, chitin varies in crystallinity, degree that a strong interaction between cationic CMs and anionic of covalent bonding to other wall components, and DD. glycosaminoglycan receptors can retain the microspheres It was reported as the principal component of protective at the target site of the capillary region. CMs have been cuticles of crustaceans such as crabs, shrimps, prawns, 9 17 10,18,19 applied in the oral, parenteral, and nasal delivery of and lobsters. encapsulated vaccine, DNA, or small interfering ribonucleic Chitosan, a natural linear polyaminosaccharide obtained 20–23 acid transfection studies. by alkaline deacetylation of chitin, is the second most abun- dant polysaccharide next to cellulose. It is made up of Biodegradability, biocompatibility, copolymers of glucosamine and N-acetyl-glucosamine, while chitin is a straight homopolymer composed of β-(1, 4)-linked and safety of CMs 13–15 N-acetyl-glucosamine units. Chitosan has one primary Biodegradability and biocompatibility play important roles amino and two free hydroxyl groups for each C6 building unit in the metabolic process of chitosan in the body. It has (Figure 1). Due to the presence of abundant amino groups, been suggested that for systemic absorption a suitable MW chitosan carries a positive charge and thus reacts with nega- (30–40 kDa) is essential for renal clearance dependent on tively charged polymers as well as with mucosal surfaces, the type of the polymer. When the size of the polymer is making it a useful polymer for mucosal delivery. Many larger than this range, then degradation is necessary for the studies have reported the use of chitosan in the formation polymer to be eliminated from the body. Degradation of of gels, nanoparticles, and microspheres for drug delivery chitosan is known to occur in vertebrates by lysosomes and 12 24 application. several bacterial enzymes. The biodegradability of chitosan submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers in living organisms is dependent on its DD of chitin wherein via the oral or nasal route is generally low. These vaccines 25,26 the degradation rate decreases with an increase in DD. are sometimes impermeable to the mucosal barrier owing to Primarily, chitosan is degraded sufficiently and eliminated their large MW and hydrophilic characteristics. Moreover, properly in most cases when given adequate conditions. they can be easily degraded by the proteolytic enzymes Chitosan, as any other drug delivery materials, should be present at the mucosal site. On the other hand, parenteral preferentially degraded after the efc fi ient delivery of vaccine injections require a relatively high dose because the in vivo to the target site. The digestion of chitosan was found to be half-life of the vaccine is generally no more than a few hours species-dependent and also dependent on the availability of which is considered one of the major problems of parenteral 27 39 the amine group in the composition of chitosan. administration. Thus, an improved system that can provide The safety of chitosan has been extensively studied and it a sustained and controlled delivery of vaccine with maximum was found that it is a biologically compatible polymer with a bioavailability is a priority. 28,29 minimal toxicity. Many countries including Japan, Italy, Chitosan is not only nontoxic and biodegradable but and Finland have approved the use of chitosan for dietary it also exhibits excellent mucoadhesive properties and application. It has also been approved by the Food and Drug permeation-enhancing effect of the delivery materials across 31 39 Administration for wound dressing application in the USA. the cell surface, especially the mucosal area. CMs also As chitosan is considered a nontoxic and nonirritant material, have potential applications for enhancing the adsorption it is widely applied as a potential excipient in pharmaceutical of mucosally administered biomacromolecules through the 40,41 formulations as well as in cosmetic industries. It is biocompat- paracellular route. They have the potential to loosen up ible for both healthy and infected skin. It has been described the tight junction between epithelial cells and to reduce that the median lethal dose for an oral administration of chitosan transepithelial electrical resistance. It is worthwhile men- in rodents was .16 g/kg, suggesting that it is safe and the tioning that mucoadhesivity is another potential benefit to risk of side effects after oral administration is negligible. On using CMs for improved drug adsorption because cationic the other hand, Dash et al found that the toxicity of chitosan chitosan interacts with the anionic mucosal layer, which has was dependent on its DD and MW. As MW and concentration sialic acid moieties. This adhesivity offers various advantages increased, the toxicity of chitosan also increased. It was noted for an enhanced uptake of the therapeutic vaccines at the that the toxicity of high DD chitosan was greatly increased by site of the induction phase: (1) mucoadhesive CMs could changes in MW and concentration when compared to that of strongly reduce degradation of the vaccine by proteases at low DD. Interestingly, chitosan and its derivatives were toxic the absorption membrane by providing an intimate interaction 33–35 to several bacteria, fungi, and parasites. This could be ben- with intestinal mucosa; (2) the adhesion of vaccine-loaded ec fi ial to controlling infectious diseases; however, the precise CMs to the mucosal layer provides an excessive driving mechanism behind this inhibitory effect is yet to be further force by a high concentration gradient towards the absorp- examined. It has been reported that no signic fi ant pyrogenic and tion membrane, leading to enhanced paracellular uptake; toxic effects of chitosan were found in mice, rabbits, and guinea and (3) the mucoadhesive properties of chitosan provides a pigs. In a fat chelation study, 4.5 g/day chitosan in humans prolonged residual time of CMs on mucosal tissue, leading was reported to be nontoxic. It was noted, however, that in to drug absorption for an extended period of time and thus 36,37 40,41 both of these studies the MW and DD were not specified. improving its bioavailability. Patil et al found a strong It has been reported that chitosan nanoparticles with 80 kDa interaction between mucin in the nasal mucus layer and CMs, MW and 80% DD showed no toxicity in mice when orally which resulted in rapid absorption and high bioavailability. delivered at 100 mg/kg. Moreover, chitosan solution exposed Moreover, CMs were cleared slowly from the nasal cavity, to nasal mucosa showed no signic fi ant changes in mucosal cell also improving bioavailability. In another study, Wang morphology compared to the control. Collectively, chitosan et al emphasized the enhancement of drug bioavailability exhibits minimal toxicity and side effects, which opens the using both the mucoadhesivity and permeation-enhancing possibility for its application and adoption in vaccine delivery effect of CMs, suggesting that CMs could not only protect as a safe and biocompatible material. vaccines from degradation but also improve permeation, uptake, and bioavailability of the drug. They further defined Bioavailability of CMs the parameters, such as size and distribution, of CMs that Most vaccines are administered by parenteral injection are important for improving drug bioavailability, reproduc- because the bioavailability of mucosally delivered vaccines ibility, and repeatability as well as steady release behavior. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress Producing equal sized CMs is very difficult, and the size Interaction with anions distribution would be too broad if the microspheres are pre- Ionotropic gelation pared by mechanical stirring or ultrasonication technique, The counterions that are used in the ionotropic gelation which are common methods for CM preparation. These method can be divided into two main categories: low could limit their vaccine delivery application. Firstly, the MW counterions (eg, pyrophosphate, tripolyphosphate, poor reproducibility of equal sized CMs may result in poor tetrapolyphosphate, octapolyphosphate, hexametaphosphate, repeatability on release behavior and efficacy among the octyl sulfate, lauryl sulfate, hexadecyl sulfate, and cetyl different batches. Secondly, the therapeutic efficacy can stearyl sulfate) and high MW counterions (eg, alginate, hardly be achieved with irregular sized CMs and a broad κ-carrageenan, and polyaldehydrocarbonic acid). Briefly, size distribution. Thirdly, a broad size distribution of CMs chitosan solution is added dropwise into magnetically stirred would result in poor bioavailability of the vaccine. Fourth, the aqueous counterions. The beads are removed from the solu- side effects of vaccine therapy would likely be increased. tion by filtration, washed with distilled water, and dried. Therefore, particle size is an important factor that should be CMs encapsulated with an atrophic rhinitis vaccine prepared taken into account in the application and pharmacodynamic by ionotropic gelation were nasally administered, which effect of vaccine-loaded CMs. Thus, it is important to prepare enhanced cytokine (tumor necrosis factor-α [TNF-α]) and CMs of uniform size with a narrow size distribution and nitric oxide production as an indication of immune stimulat- controlled release profile for their effective application in ing activity. mucosal vaccine delivery. Emulsification and ionotropic gelation Low off-target immunogenicity of CMs In the emulsification and ionotropic gelation method, an One of the major concerns of a vaccine carrier system is aqueous solution of chitosan is added to a nonaqueous the unwanted immunogenicity and pathogenicity caused continuous phase (isooctane and emulsifier) to form a water- by off-target reactions between the carrier itself and the in-oil emulsion. Sodium hydroxide solution is then added body’s immune system. This is the major disadvantage of at different intervals, leading to ionotropic gelation. The using bioengineered viruses or bacteria as delivery vehicles microspheres, thus formed, are removed by l fi tration, washed, for vaccines. Therefore, polymeric carriers have been and then dried. It has been suggested that the conventional investigated as a useful alternative for the efficient delivery emulsic fi ation and ionotropic gelation method for preparation of vaccines without unwanted immunological outcomes. of CMs provides irregular microparticles, whereas spheri- In this regard, chitosan can be considered a powerful cal microparticles with a diameter of about 10 µm can be polymer candidate because it has enormous potential for obtained when employing a modified process. In one modi - use as a vaccine carrier system that possesses low off-target fied process, gelatin is used, which allows the ionic crosslink - immunogenicity, suggesting that it will limit unwanted ing of chitosan/gelatin (water-in-oil emulsion) to take place off-target immune reactions with the body’s normal immune under coagulation conditions at a low temperature. Several function and not interfere with the actual vaccine-mediated other crosslinking agents have been used for surface modi- immune response which is to be loaded. Several reports have fication of chitosan/gelatin microspheres: the surface was also suggested that chitosan and its derivatives could be use- very smooth in sodium sulfate or sodium citrate crosslinked ful for drug delivery application without any significant off- chitosan/gelatin microspheres; however, large gaps were 47,48 target immunogenicity. Therefore, CMs (without vaccine observed in chitosan/tripolyphosphate microspheres. It has loaded) are expected to neither alter normal immunological been reported that the increase of stirring speed leads to a activity and biological function in the body nor interfere decrease in diameter and a narrower size distribution. with the vaccine efficacy by showing unwanted off-target immunogenicity. Complex coacervation Sodium alginate, sodium carboxymethyl cellulose, Preparation of CMs κ-carrageenan, and sodium polyacrylic acid can be used for Different methods have been studied and applied to pre- complex coacervation with chitosan to form microspheres pare CMs for the delivery of drugs and vaccines. Several after the interionic interaction between oppositely charged methods are discussed here in detail and are summarized polymers. For example, potassium chloride and calcium in Table 1. chloride were used to formulate the coacervate capsules of submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Table 1 Advantages and disadvantages of chitosan microspheres prepared by various methods Method Advantages Disadvantages References Interaction with anions Ionotropic gelation – These processes are simple and mild and have the following advantages: – Release of vaccine depends on various factors such as molecular 8,11,49 Emulsification and (a) use physical crosslinking by electrostatic interaction instead of weight, degree of deacetylation, and concentration of chitosan 50,51 ionotropic gelation chemical crosslinking; (b) reduce the possible toxic side effects of using and/or vaccine. Control of these factors by applying these 52,54 Complex coacervation various chemicals or reagents; (c) better control of degradation kinetics methods is sensitive to the preparation of chitosan microspheres Crosslinking with other chemicals Emulsion crosslinking – Easy to control particle size – Tedious process, uses harsh crosslinking agents 17,55,56 method – High drug loading efficiency – Crosslinking agent sometimes reacts with active agent – Controlled release with improved bioavailability – Complete removal of unreacted crosslinking agent is a challenge of this method Multiple emulsion method – Improves entrapment efficiency and loading content – Cannot avoid the use of organic solvent and crosslinking agent 12 – Better morphological characteristics – Improves production yield Thermal crosslinking method – Provides suitable particle size – Controlling the temperature is crucial because entrapment 57 efficiency and release of vaccine depends on a controlled temperature during the crosslinking process Crosslinking with a naturally – Naturally occurring agents are used as crosslinkers, thus less toxic – The physical, mechanical, and thermal stability of the 58–60 occurring agent – Smaller particle size, low crystallinity, and good sphericity microspheres prepared by this method are not well established; – Shows superior biocompatibility more investigation needed – Exhibits slow degradation rate compared to glutaraldehyde crosslinked chitosan microspheres Emulsion droplet coalescence – High loading efficiency – Particle size depends on the degree of deacetylation of chitosan. 61 method – Smaller particle size The decreased degree of deacetylation increases particle size which in turn decreases drug content Coacervation or precipitation – The process avoids the use of toxic organic solvents – Partially protects the loaded active agent from nuclease 63 method – Particle size and drug release can be controlled degradation Reverse micellar method – Smaller particle size and narrow size distribution – A tedious preparation process with many steps 64 – Thermodynamically stable particle size with suitable polydispersity index Sieving method – A simple method which is devoid of tedious processes – Irregular particle shape 65 – Can be easily scaled up Solvent evaporation method – Good entrapment efficiency and particle morphology – Controlling particle size depends on using agglomeration 66 preventing agent – Particle size decreased with the use of an increased amount of this agent Spray drying method – A popular method to prepare powder formulation – Control of size depends on size of nozzle, spray flow 67 – Good drug stability, good entrapment efficiency, and prolonged rate, pressure inlet air temperature drug release can be achieved – Entrapment efficiency depends on the molecular weight of chitosan, ie, chitosan with low molecular weight provides better entrapment efficiency Islam et al Dovepress chitosan–alginate and chitosan–κ-carrageenan, respectively, CMs had better biocompatibility and slower degradation rate 59,60 and the obtained capsules were hardened in the counterion than glutaraldehyde crosslinked CMs. The microspheres 52–54 solution before washing and drying. used as an injectable chitosan-based drug delivery system revealed low toxicity. Crosslinking methods Emulsion crosslinking method Emulsion droplet coalescence method Water insoluble reagents can be simply dispersed in chitosan Tokumitsu et al developed the emulsion droplet coalescence solution and entrapped by the emulsion crosslinking process. method for CM preparation, which implements the principle Glutaraldehyde, formaldehyde, and genipin have been widely of both emulsion crosslinking and precipitation. In this used as crosslinking agents for the preparation of CMs. In method, precipitation is usually induced by coalescence of the emulsion crosslinking method, chitosan solution is first chitosan droplets with sodium hydroxide. Briefly, a drug prepared by dissolving chitosan with acetic acid. This solu- containing stable emulsion solution of chitosan is prepared in tion is then added to liquid paraffin containing a surfactant, liquid paraffin oil. This emulsion is mixed with another s table forming a water-in-oil emulsion before the addition of a emulsion containing a chitosan aqueous solution of sodium crosslinking agent. The formed microspheres are filtered, hydroxide with high-speed stirring, which allows the drop- 17,55,56 washed with suitable solvent, and dried. lets of each emulsion to collide randomly and coalescently. This results in the precipitation of chitosan droplets with Multiple emulsion method small particle size. CMs loaded with gadopentetic acid were The multiple emulsion method is probably the best way to prepared using this method for gadolinium neutron capture increase the entrapment efficiency of the target molecule therapy. Gadopentetic acid interacts electrostatically with in CMs. In this method, a primary emulsion (oil-in-water) amino groups of chitosan since it is a bivalent anionic is first formed (nonaqueous solution containing the target compound. A range of nanosized particles and a high loading molecule in chitosan solution). This primary emulsion is of gadopentetic acid were obtained through this emulsion then added to an external oil phase to form multiple emul- droplet coalescence method compared to the conventional sions (oil-in-water-in-oil) followed by either the addition of emulsion crosslinking method. glutaraldehyde (as a crosslinking agent) or the evaporation of an organic solvent. CMs, loaded with hydrophobic reagents, Precipitation or coacervation method were found to have better morphological characteristics and Chitosan precipitates when it interacts with an alkaline yield when prepared by the multiple emulsion method. solution since it is not soluble in an alkaline pH medium. In this method, chitosan particles are prepared by dropping Thermal crosslinking method chitosan solution into an alkaline solution (eg, sodium In the thermal crosslinking technique, CMs are prepared with hydroxide, sodium hydroxide–ethanediamine, or sodium different thermal conditions in various steps. Orienti et al hydroxide–methanol) through a compressed air nozzle, reported CM preparation by the thermal crosslinking method which produces coacervate droplets. Particles are collected using citric acid, which served as crosslinking agent. Citric by precipitation or centrifugation before excessive washing acid was added to chitosan solution in acetic acid (2.5% with hot and cold water, respectively. The particle sizes weight/volume) and then cooled to 0°C before adding to can be controlled by varying the diameter of the compressed corn oil. After stirring for 2 minutes, the emulsion was then air nozzle together with the pressure. A crosslinking agent added dropwise to corn oil by maintaining the temperature can also be used to harden the particles, which would be at 120°C. Then, the crosslinking was performed under vigor- beneficial because of its slow release. Sodium sulfate was ous stirring (1000 rpm) for 40 minutes and the microspheres also used to prepare CMs using this precipitation technique. obtained were filtered, washed, dried, and sieved. Recombinant human interleukin-2-loaded CMs were pre- pared by a dropwise addition of sodium sulfate-containing Crosslinking with a naturally occurring agent recombinant human interleukin-2 solution in acidic chi- Genipin, a naturally occurring crosslinking agent, has also tosan solution. As a result, chitosan was precipitated and been used to prepare CMs by the spray drying method, recombinant human interleukin-2 was incorporated when which provides small particle size, low crystallinity, and CMs were formed. Of note, this method is devoid of any good sphericity. It was reported that genipin crosslinked crosslinking agent. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers Reversed micellar method Vaccine delivery through CMs Reverse micellar is the stable liquid mixture of oil, water, CMs have been examined for the mucosal delivery of and surfactants dissolved in organic solvents. To this mix- vaccines. A variety of chitosan-based carrier systems with ture, an aqueous solution of chitosan and the target molecule their functional properties for oral and nasal delivery is are added before the addition of a crosslinking agent such shown in Table 2. Here, the utility of CMs for oral and nasal as glutaraldehyde. Mitra et al described the preparation vaccination in vitro and in vivo are discussed. of doxorubicin–dextran conjugate-encapsulated chitosan Oral delivery nanoparticles. Oral delivery of vaccines has numerous advantages over conventional needle injection and is a well accepted route of Sieving method vaccination. However, most vaccines are still administered by Agnihotri and Aminabhavi developed a method to prepare injection due to the lack of a proper delivery system to reach the clozapine-loaded CMs. In this method, a thick jelly mass induction site and to enhance the effector responses. Although of chitosan was prepared in 4% acetic acid and crosslinked oral delivery is probably the preferred administrative route with glutaraldehyde. The crosslinked nonsticky jelly mass of vaccines, especially for children, it causes degradation of was passed through a sieve to get microparticles of a suitable the antigens in the gastrointestinal track and also shows inef- size, which were then washed with 0.1 N sodium hydroxide ficient targeting to the site of action when delivered in a naked to remove unreacted glutaraldehyde and dried overnight at form. Therefore, developing an effective delivery system 40°C. As a result, a high loading efficiency of clozapine has been considered the primary task in the oral vaccination (98.9%) was achieved. However, the particles were irregular e fi ld. To gain adequate immune responses after oral delivery, in shape with an average size of 543–698 µm. The irregular the vaccine should reach the M-cells of Peyer’s patches in shape and size of the particles is one of the major disadvan- the gut avoiding the acidic pH condition of the stomach and tages of this method, which could affect the bioavailability of enzymatic degradation. Even if the vaccine nearly reaches CMs in vivo. However, an in vitro and in vivo study demon- Peyer’s patches, the immune response is not always induced strated a controlled and sustained release of the drug. due to the inability of antigens to gain access to Peyer’s patches and because of inefc fi ient uptake at the induction site. Several Solvent evaporation method studies have shown that the uptake by M-cells was significantly The solvent evaporation method involves the formation of enhanced and degradation of protein and peptide vaccines in emulsion between a polymer solution and an immiscible the gastrointestinal track was prevented after the incorporation continuous phase – either aqueous (oil-in-water) or non- 59,68–71 of vaccine with CMs. Due to its nontoxicity and potent aqueous (water-in-oil). This can be done by using liquid antigen binding properties, chitosan has been considered a parafn fi /acetone. The target molecule dissolved in acetone is promising tool for oral vaccination. dispersed in chitosan solution and the mixture is emulsified Extensive research on CMs for mucosal vaccine delivery, in liquid paraffin while stirring. The microsphere suspen - in particular, the uptake of CMs in murine Peyer’s patches sion is filtered, washed, and dried. Magnesium stearate can in vitro and in vivo, was carried out by van der Lubben be added as an agglomeration preventing agent. It appears 59,68,70,71 et al. They prepared a human intestinal M-cell model that the average particle size decreases when the amount of by coculturing Caco-2 and Raji-cells and investigated the magnesium stearate used in the preparation is increased. uptake of CMs. No morphological changes in the mono- Spray drying method layer were observed and this model was used to examine Spray drying is one of the most widely investigated methods the in vitro uptake of CMs for oral vaccine delivery. They of preparing CMs in which chitosan solution is sprayed and found that CMs can be taken up by the epithelium of Peyer’s then air-dried followed by the addition of a crosslinking patches. It has been reported that the size of microparticles agent. He et al prepared CMs by spray drying multiple emul- should be ,10 µm for efficient uptake by M-cells and to sions (oil-in-water-in-oil or water-in-oil-in-water) to entrap reach the dome of Peyer’s patches. Indeed, CMs used in the cimetidine and famotidine into microspheres. The drug was study were much smaller than 10 µm and therefore suitable released in a sustained and controlled fashion compared to for M-cell uptake. Since chitosan is biodegradable, van der the other microspheres prepared by traditional spray drying Lubben et al further claimed that antigen was freed from CMs 67 68 or the oil-in-water emulsion method. after uptake by M-cells. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress fi submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Table 2 Chitosan-based carrier systems with functional properties for the delivery of (model) vaccines through oral and nasal routes Chitosan-based carrier type Delivery route (Model) vaccines Functional properties References Chitosan microparticles Oral Ovalbumin – Targets Peyer’s patches for M-cell uptake 59,68 Chitosan microparticles Oral Tetanus toxoid – Strong systemic and local immune responses 69 Chitosan microparticles Oral/ Diphtheria toxoid – Enhancement of both systemic and local immune responses 70 nasal Eudragit -coated chitosan Oral Ovalbumin – A controlled release profile of drug from the microspheres toward Peyer’s patches 72 microspheres – Induces proper immune stimulation Thiolated Eudragit-coated Oral Bovine serum albumin – Retains structural integrity of protein 4 chitosan microspheres – Improvement of mucoadhesiveness and residual time at the target site Chitosan microspheres (mixed Oral Hepatitis B surface antigen – Enhancement of antigen stability 73 with protease inhibitors – Strategic potential against chronic hepatitis B and permeation enhancer) Albumin–chitosan mixed matrix Oral Typhoid vi antigen – Induction of antigen-specific systemic and mucosal immune response 74 microspheres Chitosan microspheres Nasal Bordetella bronchiseptica – Shows suitable, but with some aggregation, physicochemical properties 49 dermonecrotoxin – Enhances immune stimulating activity in vitro and in vivo Pegylated chitosan microspheres Nasal B. bronchiseptica dermonecrotoxin – Improves stability and avoids aggregation of the microspheres 92 – Improvises immune stimulatory activity compared to chitosan microspheres alone Mannosylated chitosan Nasal B. bronchiseptica dermonecrotoxin – Specifically targets macrophages through the mannosylated moieties of mannose 91 microspheres receptor on the cell surface – Increases immune stimulatory activity in vitro and in vivo through specic targeting and activation of macrophages Chitosan microspheres Nasal N/A – No perceptible toxic effects 95,96 or chitosan solution alone – Increases bioadhesive properties Heat-labile toxin formulated Nasal LTK63 mutant of heat-labile – Induces high antigen-specific systemic and mucosal immune response 88,97 chitosan or N-trimethyl toxin (as adjuvant) chitosan microspheres Chitosan–DNA nanospheres Nasal DNA encoding Respiratory – Strong cell-mediated immune response 98 syncytial viral antigens Antigen-loaded chitosan/ Nasal Tetanus toxoid, diphtheria toxoid, – Stabilization of protein antigen by F127 105,106 Pluronic F127 microparticles and anthrax recombinant protective – Antigen stabilization strongly enhances the systemic and mucosal immune antigen response of chitosan/F127 than that of chitosan microparticles alone Dovepress Chitosan microspheres as vaccine carriers Therapeutic use of CMs for oral and nasal delivery protect chitosan from the acidic stomach. When this reaches has been examined. A diphtheria toxoid (DT) was used the intestine, the enteric layer dissolves at high pH and the to examine the enhancement of both systemic and local antigen-encapsulated chitosan core is exposed to enzymes. immune responses. Unloaded CMs, DT-loaded CMs, and In this state, chitosan can protect the encapsulated antigen DT in phosphate-buffered saline (PBS) were delivered into from enzymatic degradation and most importantly can lead mice by oral and nasal administration. DT associated with the antigen to reach the induction site of Peyer’s patches for alum was subcutaneously immunized in mice as a positive immune stimulation. For this, Hori et al developed Eudragit - control. A strong systemic and local immune response was coated CMs and evaluated ovalbumin as an oral immune found against DT in mice administered orally with different delivery system. The ovalbumin-loaded CMs prepared doses of DT-loaded CMs when compared to the mice fed by the emulsification-solvent evaporation method showed with DT in PBS. Furthermore, a dose-dependent anti-DT high ovalbumin content and an appropriate size for the immunoglobulin G (IgG) response in sera was found after efficient uptake by Peyer’s patches. A comparable systemic oral administration of DT-loaded CMs. On the other hand, the IgG response was found after the oral administration of systemic immune response (IgG) induced by DT-associated ovalbumin-loaded CMs in mice. Moreover, a higher intestinal CMs were ten times higher than that induced with DT in PBS mucosal IgA response was achieved using ovalbumin-loaded after nasal delivery. CMs by delivery of the microspheres toward Peyer’s patches, CMs were also examined after oral delivery of tetanus where they were subsequently uptaken by the M-cells and the 69 72 toxoid (TT) to induce systemic and local immune responses. entrapped ovalbumin was released in a controlled fashion. TT-loaded CMs were prepared by the ionic crosslinking In another study, Cho et al reported a mucoadhesive and pH- method using sodium tripolyphosphate. Unloaded CMs, sensitive thiolated Eudragit-coated CM, designed to enhance TT-loaded CMs, and naked TT in PBS were orally adminis- mucoadhesivity and bioavailability of the carrier at the target tered in mice, and TT absorbed on aluminum phosphate was site. They found strong mucoadhesive properties in vitro administered intramuscularly as a positive control. TT-loaded and in vivo, suggesting that Eudragit-coated CMs were a CMs enhanced a strong systemic and local immune response potential carrier for the oral delivery of vaccines. in a dose-dependent manner at 3 weeks after the oral delivery Recently, hepatitis B surface antigen-loaded CMs were of vaccine compared to TT in PBS. They observed that a formulated, characterized, and optimized in vitro and in vivo four-fold higher dose was needed for TT-loaded CMs to get for effective oral delivery of hepatitis B surface antigen a similar IgG response to the positive control. The study was against chronic hepatitis B. An emulsion solvent evapora- also carried out at different time points to understand the tion technique was applied to prepare CMs, with the addition kinetics of the immune response based on the level of IgG. It of protease inhibitors and permeation enhancers to overcome was found that the IgG response could be observed at day 14 the limitation of the enzymatic and permeation barrier. and was increased after boosting at day 22. At day 29, the IgG In vitro drug release, in vivo efficacy, and importantly the level was lower than at day 22; however, it still maintained effect of different storage conditions were studied to test a higher concentration than TT in PBS at all the time points the practicality of the system. An enhanced stability of the investigated. On the other hand, IgA levels were not sig- antigen was found when using the microspheres for a period nificantly different at day four; however, the levels were of 4 months at room temperature, suggesting a possible way significantly ( P , 0.01) higher in TT-loaded CMs than in TT to overcome the tedious and expensive requirement of cold in PBS at days eight, 14, and 22. These results suggest that chain storage in the vaccine industry. Importantly, the study the encapsulated vaccine in CMs enhanced the systemic as signifies a potential strategy for effective oral administration well as local immune responses compared to the nonencap- of hepatitis B surface antigen using the biodegradable CM sulated vaccine, rendering a safe and effective form of oral system. vaccination. Further studies on cellular immune responses Recently, Uddin et al developed an albumin–chitosan including memory effect of B-cells and T-cells will ensure mixed matrix microsphere (ACM)-l fi led capsule formulation the solid effectiveness of CMs for vaccine delivery. for oral administration of Typhoid Vi antigen (TVA) to At first glance, chitosan would not be considered sui t- demonstrate antigen-specic fi systemic and mucosal immune able for oral vaccination since it is a pH-sensitive polymer. responses. TVA-loaded ACMs were filled into hard gelatin It is soluble at acidic pH and becomes insoluble at about capsules with enteric coating. The physicochemical char- pH 6.5. It has been suggested that an enteric coating can acterization such as particle size, zeta potential, swelling, submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress and disintegration rates of the microspheres were favorable and a major virulence factor for atrophic rhinitis – a disease for oral delivery of the microencapsulated vaccine. In vivo that causes huge economic damage in the swine industry. studies showed that the oral delivery of TVA-loaded ACMs BBD-loaded CMs were prepared by the ionic gelation process had similar IgG and IgA responses with those of the par- using tripolyphosphate. The morphology of vaccine-loaded enteral vaccination group, suggesting that TVA-loaded microspheres was observed as aggregated shapes, whereas ACMs had the potential to induce antigen-specific immune unloaded microspheres were quite spherical. The average responses when delivered via oral administration. particle size of BBD-loaded CMs was 4.39 µm, which provides a condition for effective delivery of the vaccine Nasal delivery to nasal-associated lymphoid tissue for immune induction. Nasal administration of vaccines has been reported to The size of unloaded CMs was about 1.94 µm, indicating enhance bioavailability and improve efficacy. An effec- that CMs became enlarged after vaccine loading. The release tive humoral and cell-mediated immune response can be studies further demonstrated that when the MW of chitosan achieved through nasal delivery of vaccines when the decreased, more BBD was released. It was also found that appropriate delivery system is used as a carrier for particu- encapsulated BBD had greater release at higher pH than lower 75,76 late antigens. Nasal- associated lymphoid tissue, present pH. The secretion of TNF-α and nitric oxide from the murine at the nasal epithelium and containing immunocompetent macrophages treated with BBD-loaded CMs indicated that cells, would be an ideal target site for the nasal delivery of the cells stimulated with BBD-loaded CMs produced TNF-α 75,76 vaccines to induce an immune response. It has been sug- and nitric oxide in a time-dependent manner at a similar gested that nasal- associated lymphoid tissue epithelium has level to cells stimulated with BBD alone or lipopolysac- similar types of immune cells that are present in the M-cells charide. It is important to mention that BBD-loaded CMs of Peyer’s patches in gut-associated lymphoid tissue and induced a steadily increasing immune stimulating effect in is located just below the epithelial surface, which contains the macrophages, whereas it began to decrease at 80 hours macrophages, dendritic cells, lymphoid follicles (mostly poststimulation with lipopolysaccharide. B-cells), and intrafollicular areas (mostly T-cells) in a An in vivo study was carried out in mice that measured network. At these sites, particulate antigens are mainly taken IgG and IgA in sera, nasal wash, and saliva after intranasal up and/or transported across the cells by transcytosis without administration of BBD-loaded CMs. The IgA levels in nasal any extensive degradation. It has been well described that wash increased in a time- and dose-dependent manner after increased epithelial permeability influences the particulate intranasal administration of BBD-loaded CMs. However, such 77–84 antigen uptake across the epithelial mucosa. Importantly, immune response was not detected in saliva, suggesting that chitosan has the ability to increase membrane permeability CMs successfully delivered the vaccine to nasal-associated when used as a delivery system for nasal vaccination. lymphoid tissue after intranasal administration and induced However, antigen delivery through nasal administration a higher systemic and local immune response. Although in sometimes results in poor immune responses. Several factors vitro and in vivo results showed CMs as a potential carrier including limited diffusion of particulate antigens across the for nasal delivery, BBD-loaded CMs were found in aggre- mucosal barrier, rapid clearance of particulate drug or vac- gated shapes because of physical and storage instabilities. cine formulation from the mucosal surface, and enzymatic To overcome this instability problem, chitosan was degradation because of instability of the particulate carrier are modified by covalent conjugation with polyethylene glycol 86,87 92 associated with this. In order to overcome these problems, to form pegylated chitosan. The pegylated CMs (PCMs) chitosan might be one of the best options for nasal administra- were prepared through a similar ionic gelation process. The tion of vaccines due to its ability to increase the retention time average particle size of BBD-loaded PCMs was ,10 µm, when it binds to the mucosal membrane. Several reports their shape was spherical, and they were physically more also demonstrated that chitosan enhanced mucosal absorp- stable compared to BBD-loaded CMs. Due to better sta- tion of vaccines with adjuvant activity to improve mucosal bility, the vaccine was released from BBD-loaded PCMs 10,88,89 immunity after nasal administration. in a more steady fashion than in BBD-loaded CMs. The Cho and colleagues conducted extensive research on CMs study further showed that macrophages secreted TNF-α for intranasal delivery of vaccines to induce the immune and nitric oxide in a time-dependent manner after expo- 49,90–94 response in vitro and in vivo. They used Bordetella sure to BBD, BBD-loaded CMs, BBD-loaded PCMs, and bronchiseptica dermonecrotoxin (BBD), a causative agent lipopolysaccharide. However, a significantly higher TNF- α submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers secretion was found in the cells treated with BBD-loaded used as a nasal vaccine delivery carrier without any harmful 95,96 PCMs than cells exposed to BBD-loaded CMs and BBD effects. To investigate this, the cilia beat frequency was alone. Moreover, TNF-α secretion increased in a sus- studied in guinea pigs after nasal administration of chitosan tained fashion in the cells exposed to BBD-loaded PCMs, solution for 28 days and found that none of the chitosan whereas it began to decline at 48 hours poststimulation induced the changes of cilia beat frequency, indicating a 92 95 with lipopolysaccharide. safety profile of chitosan for nasal delivery. They further To increase the target specificity, another study was investigated the bioadhesive properties of CMs via nasal carried out with mannosylated CMs (MCMs) with encap- administration using three different formulations: chitosan sulated BBD to target macrophage mannose receptors and solution, CMs, and starch microspheres, which was fol- increase immune stimulating activity. Colocalization of lowed by the examination of clearance properties in human BBD-loaded MCMs and the macrophage receptors was con- subjects. The clearance rate was 21 minutes for the control, firmed by confocal laser scanning microscope. The results 41 minutes for the chitosan solution, 68 minutes for the showed that macrophages exposed to BBD-loaded MCMs starch microspheres, and 84 minutes for the CMs. This secreted higher TNF-α and interleukin-6 than that of BBD- result indicates that CMs have better bioadhesive proper- loaded CMs and BBD alone. Furthermore, BBD-specific ties and are able to significantly reduce the drug clearance IgA response was found to be significantly higher in saliva rate and prolong the residence time of the delivered vaccine and serum after intranasal immunization with BBD-loaded in nasal mucosa, resulting in enhanced bioavailability and 91 96 MCMs in mice compared with BBD-loaded CMs, sug- efficacy. gesting that the MCMs extensively assisted in stimulating Several reports demonstrated the concomitant use macrophages for induction and enhancement of immune of CMs as a mucosal adjuvant and as a vaccine delivery activity. The representative scanning electron microscope system. A vaccine formulation with CMs and a nontoxic photographs of CMs and MCMs (BBD loaded and unloaded) LTK63 mutant of heat-labile toxin induced significantly are shown in Figure 2. higher IgG titers in sera and IgA in nasal washes after intra- Soane et al performed extensive research using differ- nasal delivery in mice. A modified N-trimethyl chitosan ent types of chitosan and concluded that chitosan could be microparticulate system also showed higher antigen-specific antibody responses in sera, nasal, and vaginal wash. Chitosan–DNA nanospheres with intranasal delivery exhib- ited significant responses of cytotoxic T-cell response and interferon-γ as well as antigen specific-IgG and IgA, render - ing a strong humoral and cell-mediated immune response. CMs were prepared with Pluronic F127 as an immuno- modulating and stabilizing agent to enhance the stability for controlled drug release and adjuvanticity. Pluronic, a triblock 5 µm 5 µm copolymer of polyethylene oxide and polypropylene oxide CMs BBD-CMs (polyethylene oxide-b-polypropylene oxide-b- polyethylene oxide) commonly known as poloxamer, has a variety of pharmaceutical applications and has become one of the most extensively investigated temperature-sensitive materials. F127 is water soluble and has a good drug release profile, which makes it a potent drug delivery carrier for a variety 100–104 of therapeutic and bioactive agents. When Westerink 5 µm 5 µm et al intranasally immunized antigen-loaded F127/CMs into MCMs BBD-MCMs mice, it signic fi antly increased systemic and mucosal immune Figure 2 Scanning electron microscope photographs of CMs, BBD-loaded CMs, responses compared to those of control groups, suggesting MCMs, and BBD-loaded MCMs (5000×). Notes: Bar represents 5 µm. Reprinted from Biomaterials, 29(12). Jiang HL, Kang ML, that the stabilization of protein antigens by F127 enhances the Quan JS, et al. The potential of mannosylated chitosan microspheres to target immune response of F127/CMs compared to chitosan alone. macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization, 1931–1939. Copyright 2008 with permission from Elsevier. This study demonstrated a nasal vaccine delivery strategy Abbreviations: BBD, Bordetella bronchiseptica dermonecrotoxin; CM, chitosan microsphere; MCM, mannosylated chitosan microsphere. for enhancement of the immune response via a synergistic submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress effect of chitosan and F127. In another study, intraperitone- chitosan (eg, thiolated chitosan) might improve the stability ally and subcutaneously injected F127/cytosine–phosphate– and functionality of CMs. guanosine and F127/CM formulations signic fi antly enhanced antigen-specic fi systemic antibody responses compared to the Thiolated CMs as a modified antigens delivered with cytosine–phosphate–guanosine or and improved form of a chitosan- CMs alone, suggesting that F127 might have an adjuvant based mucosal vaccine carrier effect when used in combination with chitosan. Therefore, Thiolated polymers (ie, thiomers) have gained considerable application of a delivery system that combines adjuvants attention – especially for vaccine delivery – because they are with various modes of action is beneficial to maximizing one of the most promising polymers with multifunctional immune response. properties including strong mucoadhesivity, enhanced per- meation effects, protection ability, stability, and enhanced Limitations of CMs 109–114 bioavailability of drugs. Among various thiomer-based Besides the enormous advantages of CMs such as biode- carriers, thiolated CMs (TCMs) are highly popular because gradability, nontoxicity, permeation enhancing effects, and of their strong mucoadhesiveness and ability to control an ability to open the tight junction between epithelial cells and extend drug release profiles with improved permeation as described earlier, there are some limitations as well. 115–119 ability. TCMs can be prepared by immobilizing the Cho and colleagues performed several studies on CMs for thiol-bearing chain on the polymeric backbone of chitosan 49,90–94 vaccine delivery. They found that the vaccine-loaded (Figure 3). The strong mucoadhesivity of TCMs is obtained CMs self-aggregated at 2 weeks after preparation, although through the formation of disulfide bonds between the thiol it was effective in inducing immune responses including groups of TCMs and cysteine-rich subdomains of mucin cytokine expression in vitro and antigen-specic fi IgG and IgA glycoproteins at the mucosal surface (Figure 4). The per- 49,90 responses in vivo after nasal delivery. To make stable and meability through the mucosal surface can be enhanced by nonaggregated CMs, they used F127 to prepare F127/CMs which showed spherical morphology with no aggregation at an extended period of time after preparation. This was due CH OH CH OH CH OH 2 2 O O O to the hydrophilic polyethylene oxide chains of F127 that OH O O hindered the self-aggregation of CMs. F127/CMs showed OH OH OH OH much improved immune activity in vitro and in vivo and also NH NH NH exhibited potential protection against infection compared to 2 n 2 CMs alone. X Several other studies described the instability of CMs in SH acidic media, especially when prepared by the precipitation Thiolated chitosan method. CMs prepared by sodium sulfate precipitation were found to have poor acidic stability. This acidic instability was initiated by the addition of sodium sulfate to chitosan CH OH CH OH CH OH acetic acid solution which led to an ionic neutralization of O O O OH the positively charged amine groups of chitosan, providing O O OH OH OH poorly soluble chitosan derivatives. After the addition OH of acid (increasing proton concentration), the equilibrium NH NH NH 2 2 shifted to the solubilizing range for chitosan, thus dissolving the CMs. In another study, sulfadiazine-loaded chitosan NH beads were prepared using tripolyphosphate; however, it SH was found that the beads had poor mechanical strength. Collectively, there are some limitations of CMs that can Chitosan-N-acetyl-cysteine be overcome by modifying the CMs. For example, F127 Figure 3 Representative structure of thiolated chitosan: (A) general structure of is a good strategy to improve the stability and mechanical thiolated chitosan modified by an –SH group (X: linker) and (B) chitosan-N-acetyl- strength of CMs. Additionally, structural modifications of cysteine (modification of chitosan at the D-glucosamine unit by N-acetyl-cysteine). submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Mucin Mucin Mucin Mucin Mucin Mucin Dovepress Chitosan microspheres as vaccine carriers acid, thioglycolic acid, glutathione, and 2-iminothiolane are SH Mucin SH the aliphatic thiol-bearing ligands with functional carboxyl SH SH SH + Mucin TC TC + groups which form amide bonds with the amino groups of SH TC SH TC chitosan by carbodiimide to synthesize the thiomers of chi- SH TC Mucin TC SH SH + Vaccine 118,121–125 TC + tosan. CMs prepared by these thiomers exhibit strong TC SH Mucin TC + mucoadhesivity, biocompatibility, and enhanced permeability TC TC SH SH + TC + SH Mucin and absorption after oral and nasal administration. SH SH SH It is important to note that thiomers bearing free thiol SH Mucin groups are relatively unstable in solution because they are prone to oxidize at pH $ 5, leading to a self-crosslinking of the polymer. Different approaches have been attempted to delay oxidation and inhibit the self-crosslinking reaction. As an example of a next-generation thiomer, the aromatic thiol-bearing ligands are extraordinary candidates for delay- ing the oxidation process and protecting the thiol groups of the thiolated polymers. Recently, Bernkop-Schnurch et al TC TC + performed several studies using aromatic thiol-bearing ligands TC TC TC for the synthesis of S-protected thiolated chitosan and evalu- TC Vaccine + 109,127,128 TC ated their efficacy as mucosal drug delivery carriers. + TC TC TC TC To prepare the S-protected thiolated chitosan, the thiol-bearing TC + ligand was covalently attached to chitosan as the first step of modic fi ation. In the second step, the thiol group of thiolated chitosan was protected by the formation of disulfide bonds with aromatic thiol-bearing ligands. The S-protected thio- lated chitosan exhibited improved mucoadhesivity, enhanced permeation effect, inhibited efu fl x pump, bioavailability, and Thiolated chitosan SH Thiol group TC controlled release profile compared to the corresponding Thiolated chitosan 109,127,128 Mucin glycoprotein TCMs thiolated and unmodified polymers, demonstrating Mucin microspheres at mucus layer that TCMs prepared using S-protected thiolated chitosan are Disulfide bond a promising chitosan-based mucoadhesive polymer for the development of various mucosal vaccine delivery systems. Figure 4 Schematic representation of functional interaction between TCMs and mucin in mucosal vaccine delivery. Abbreviation: TCM, thiolated chitosan microsphere. Conclusion and future perspectives Among various investigated vaccine carriers, CMs hold using TCMs instead of unmodified CMs. Increased perme - enormous promise as a delivery vehicle for both oral and ability is achieved by opening the tight junction after the nasal administration. This review has discussed and evalu- inhibition of protein tyrosine phosphatase, a key enzyme ated various methods for preparation of CMs which could involved in the closing process of tight junction. Due to help to design more and better functionalized chitosan-based the formation of inter- and intramolecular disulfide bonds carrier systems. This study demonstrated that vaccine-loaded through TCMs, a compact three-dimensional network is CMs could be prepared with suitable and appropriate particle generated which allows controlled drug release and leads to sizes, which is a very important factor in the delivery of the high cohesivity. Moreover, TCMs exhibit a reversible opening vaccine to the induction site of mucosa-associated lymphoid of the tight junction, which leads to better permeation effects tissue for proper immune stimulation. Furthermore, both 115,117,120 than unmodified CMs. In the case of first-generation systemic and local immune responses can be induced in a thiomers, thiolated chitosan derivatives are prepared by con- dose- and time-dependent manner through vaccine-loaded jugating thiol-bearing aliphatic ligands to the amino groups of CMs. The nontoxic, highly bioavailable, mucoadhesive, chitosan. For example, N-acetyl-cysteine, 6-mecaptonicotinic and biodegradable nature of chitosan and its particulate submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Mucin Mucin Mucin Mucin Mucin Mucin Islam et al Dovepress 6. Eyles JE, Sharp GJ, Williamson ED, Spiers ID, Alpar HO. Intra nasal form is the main reason that it could become a successful administration of poly-lactic acid microsphere co-encapsulated Yersinia vaccine carrier in the near future. Furthermore, the much pestis subunits confers protection from pneumonic plague in the mouse. improved properties of modified CMs (eg, TCMs), such as Vaccine. 1998;16(7):698–707. 7. Janes KA, Calvo P, Alonso MJ. Polysaccharide colloidal par- increased mucoadhesivity, membrane permeability, stability, ticles as delivery systems for macromolecules. Adv Drug Deliv Rev. and controlled/extended release of the encapsulated vac- 2001;47(1):83–97. 8. Mi FL, Shyu SS, Chen CT, Schoung JY. Porous chitosan microsphere cine, show that they are a promising candidate for a potent for controlling the antigen release of Newcastle disease vaccine: vaccine carrier system. 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Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review

Islam, Mohammad Ariful; Firdous, Jannatul; Choi, Yun-Jaie; Yun, Cheol-Heui; Cho, Chong-Su
International Journal of Nanomedicine , Volume 7 – Dec 13, 2012
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Pubmed Central
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© 2012 Islam et al, publisher and licensee Dove Medical Press Ltd.
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1176-9114
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1178-2013
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10.2147/IJN.S38330
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Abstract

International Journal of Nanomedicine Dovepress open access to scientific and medical research Open Access Full Text Article R E v IEW Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review 1–3, Mohammad Ariful Islam * Abstract: Chitosan, a natural biodegradable polymer, is of great interest in biomedical research 1–3, due to its excellent properties including bioavailability, nontoxicity, high charge density, and Jannatul Firdous * mucoadhesivity, which creates immense potential for various pharmaceutical applications. It Yun-Jaie Choi 1–4 has gelling properties when it interacts with counterions such as sulfates or polyphosphates Cheol-Heui Yun and when it crosslinks with glutaraldehyde. This characteristic facilitates its usefulness in the 1,2 Chong-Su Cho coating or entrapment of biochemicals, drugs, antigenic molecules as a vaccine candidate, Department of Agricultural and microorganisms. Therefore, chitosan together with the advance of nanotechnology can Biotechnology, Research Institute for be effectively applied as a carrier system for vaccine delivery. In fact, chitosan microspheres Agriculture and Life Sciences, Center for Food and Bioconvergence, World have been studied as a promising carrier system for mucosal vaccination, especially via the Class University Biomodulation oral and nasal route to induce enhanced immune responses. Moreover, the thiolated form of Program, Seoul National University, Seoul, South Korea chitosan is of considerable interest due to its improved mucoadhesivity, permeability, stability, and controlled/extended release profile. This review describes the various methods used to *These authors contributed equally to this work design and synthesize chitosan microspheres and recent updates on their potential applications for oral and nasal delivery of vaccines. The potential use of thiolated chitosan microspheres as next-generation mucosal vaccine carriers is also discussed. Keywords: chitosan microspheres, oral, nasal, vaccine delivery, mucosal and systemic immune responses Introduction Vaccination is cost-effective, and probably the best preventable strategy against most diseases. Traditionally, vaccines are administered parenterally via an intramuscular 2,3 or subcutaneous route. This process of vaccine delivery incurs difficulties such as needle phobia, low patient compliance, short half-life, potential contamination while using needles, and a necessity for highly trained personnel. As a result, oral and nasal vaccination has been paid considerable attention as a way to overcome such potential drawbacks and eliminate the problems associated with parenteral administration of vac- Correspondence: Cheol-Heui Yun; cines. Better yet, parenteral vaccination mostly stimulates systemic immunity, whereas Chong-Su Cho mucosal vaccination tends to confer both systemic and mucosal immune responses. Department of Agricultural Biotechnology and Research Institute In regard to mucosal administration of protein drugs or vaccines, microspheres are for Agriculture and Life Sciences, 6–8 well known for their controlled delivery formulation, which would provide a long- Seoul National University, 1 Gwanak-ro, Gwanak-gu, lasting boosting effect and enhance the effectiveness of the immune response against Seoul 151-921, South Korea infectious diseases. Tel +82 2 880 4802 (CHY); +82 2 880 4868 (CSC) Chitosan has well-defined properties including bioavailability, biocompatibility, Fax +82 2 875 2494 (CSC) low cost, and an ability to open the intracellular tight junction; therefore, it has been Email [email protected] (CHY); [email protected] (CSC) suggested as a suitable polymeric material for mucosal delivery. Desirable properties submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 6077–6093 Dovepress © 2012 Islam et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article http://dx.doi.org/10.2147/IJN.S38330 which permits unrestricted noncommercial use, provided the original work is properly cited. Islam et al Dovepress of chitosan can be determined from its molecular weight CH (MW) and degree of deacetylation (DD). It has been reported O that high MW chitosan enhances the absorption of various NH CH OH 9,10 compounds across the mucosal barrier. Due to its cationic O O OH property, positively charged chitosan would have an elec- OH trostatic interaction with the negatively charged mucosal surface. Moreover, chitosan possesses mucoadhesivity, CH OH NH beneficial for prolonging the retention time at the mucosal area for a controlled and sustained therapeutic effect. CH n Nontoxicity is another prerequisite property of chitosan, Chitin which can be effectively applied for mucosal delivery of vaccines as a form of the microparticulate system. In an CH OH CH OH CH OH aqueous environment, chitosan swells and forms a gel- 2 2 O O O like layer, favorable for the interaction of polymers with OH O O glycoprotein in mucous. In the case of nasal delivery, OH OH OH OH chitosan possesses good bioadhesive properties and can NH NH NH 2 2 n reduce the rapid clearance of vaccine from the nasal cavity 2 where it could be delivered to nasal-associated lymphoid tissue – the induction and effector sites for vaccine-induced Chitosan immune responses. Figure 1 Structure of chitin and chitosan. General aspects of chitin Chitosan microspheres (CMs) and chitosan Extensive research has been carried out to exploit the use of Chitin is an abundant source of chitosan, a unique cationic chitosan as a drug or vaccine carrier. Indeed, chitosan has p o ly s a c c h a r i d e s u p e r i o r t o a ny m a n - m a d e c a t i o n i c been used for prolonged and targeted delivery of drug and derivatives. In general, it comprises the skeletal materials macromolecules. CMs can be a better option due to their in invertebrates. It is also found in egg shells of nematodes ability for sustained release and improved bioavailability of and rotifer as well as in the cuticles of arthropods, exo- target molecules. CMs also enhance the uptake of hydrophilic skeletons, peritrophic membranes, and cocoons of insects. substances across epithelial cells. It has been reported In the fungal walls, chitin varies in crystallinity, degree that a strong interaction between cationic CMs and anionic of covalent bonding to other wall components, and DD. glycosaminoglycan receptors can retain the microspheres It was reported as the principal component of protective at the target site of the capillary region. CMs have been cuticles of crustaceans such as crabs, shrimps, prawns, 9 17 10,18,19 applied in the oral, parenteral, and nasal delivery of and lobsters. encapsulated vaccine, DNA, or small interfering ribonucleic Chitosan, a natural linear polyaminosaccharide obtained 20–23 acid transfection studies. by alkaline deacetylation of chitin, is the second most abun- dant polysaccharide next to cellulose. It is made up of Biodegradability, biocompatibility, copolymers of glucosamine and N-acetyl-glucosamine, while chitin is a straight homopolymer composed of β-(1, 4)-linked and safety of CMs 13–15 N-acetyl-glucosamine units. Chitosan has one primary Biodegradability and biocompatibility play important roles amino and two free hydroxyl groups for each C6 building unit in the metabolic process of chitosan in the body. It has (Figure 1). Due to the presence of abundant amino groups, been suggested that for systemic absorption a suitable MW chitosan carries a positive charge and thus reacts with nega- (30–40 kDa) is essential for renal clearance dependent on tively charged polymers as well as with mucosal surfaces, the type of the polymer. When the size of the polymer is making it a useful polymer for mucosal delivery. Many larger than this range, then degradation is necessary for the studies have reported the use of chitosan in the formation polymer to be eliminated from the body. Degradation of of gels, nanoparticles, and microspheres for drug delivery chitosan is known to occur in vertebrates by lysosomes and 12 24 application. several bacterial enzymes. The biodegradability of chitosan submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers in living organisms is dependent on its DD of chitin wherein via the oral or nasal route is generally low. These vaccines 25,26 the degradation rate decreases with an increase in DD. are sometimes impermeable to the mucosal barrier owing to Primarily, chitosan is degraded sufficiently and eliminated their large MW and hydrophilic characteristics. Moreover, properly in most cases when given adequate conditions. they can be easily degraded by the proteolytic enzymes Chitosan, as any other drug delivery materials, should be present at the mucosal site. On the other hand, parenteral preferentially degraded after the efc fi ient delivery of vaccine injections require a relatively high dose because the in vivo to the target site. The digestion of chitosan was found to be half-life of the vaccine is generally no more than a few hours species-dependent and also dependent on the availability of which is considered one of the major problems of parenteral 27 39 the amine group in the composition of chitosan. administration. Thus, an improved system that can provide The safety of chitosan has been extensively studied and it a sustained and controlled delivery of vaccine with maximum was found that it is a biologically compatible polymer with a bioavailability is a priority. 28,29 minimal toxicity. Many countries including Japan, Italy, Chitosan is not only nontoxic and biodegradable but and Finland have approved the use of chitosan for dietary it also exhibits excellent mucoadhesive properties and application. It has also been approved by the Food and Drug permeation-enhancing effect of the delivery materials across 31 39 Administration for wound dressing application in the USA. the cell surface, especially the mucosal area. CMs also As chitosan is considered a nontoxic and nonirritant material, have potential applications for enhancing the adsorption it is widely applied as a potential excipient in pharmaceutical of mucosally administered biomacromolecules through the 40,41 formulations as well as in cosmetic industries. It is biocompat- paracellular route. They have the potential to loosen up ible for both healthy and infected skin. It has been described the tight junction between epithelial cells and to reduce that the median lethal dose for an oral administration of chitosan transepithelial electrical resistance. It is worthwhile men- in rodents was .16 g/kg, suggesting that it is safe and the tioning that mucoadhesivity is another potential benefit to risk of side effects after oral administration is negligible. On using CMs for improved drug adsorption because cationic the other hand, Dash et al found that the toxicity of chitosan chitosan interacts with the anionic mucosal layer, which has was dependent on its DD and MW. As MW and concentration sialic acid moieties. This adhesivity offers various advantages increased, the toxicity of chitosan also increased. It was noted for an enhanced uptake of the therapeutic vaccines at the that the toxicity of high DD chitosan was greatly increased by site of the induction phase: (1) mucoadhesive CMs could changes in MW and concentration when compared to that of strongly reduce degradation of the vaccine by proteases at low DD. Interestingly, chitosan and its derivatives were toxic the absorption membrane by providing an intimate interaction 33–35 to several bacteria, fungi, and parasites. This could be ben- with intestinal mucosa; (2) the adhesion of vaccine-loaded ec fi ial to controlling infectious diseases; however, the precise CMs to the mucosal layer provides an excessive driving mechanism behind this inhibitory effect is yet to be further force by a high concentration gradient towards the absorp- examined. It has been reported that no signic fi ant pyrogenic and tion membrane, leading to enhanced paracellular uptake; toxic effects of chitosan were found in mice, rabbits, and guinea and (3) the mucoadhesive properties of chitosan provides a pigs. In a fat chelation study, 4.5 g/day chitosan in humans prolonged residual time of CMs on mucosal tissue, leading was reported to be nontoxic. It was noted, however, that in to drug absorption for an extended period of time and thus 36,37 40,41 both of these studies the MW and DD were not specified. improving its bioavailability. Patil et al found a strong It has been reported that chitosan nanoparticles with 80 kDa interaction between mucin in the nasal mucus layer and CMs, MW and 80% DD showed no toxicity in mice when orally which resulted in rapid absorption and high bioavailability. delivered at 100 mg/kg. Moreover, chitosan solution exposed Moreover, CMs were cleared slowly from the nasal cavity, to nasal mucosa showed no signic fi ant changes in mucosal cell also improving bioavailability. In another study, Wang morphology compared to the control. Collectively, chitosan et al emphasized the enhancement of drug bioavailability exhibits minimal toxicity and side effects, which opens the using both the mucoadhesivity and permeation-enhancing possibility for its application and adoption in vaccine delivery effect of CMs, suggesting that CMs could not only protect as a safe and biocompatible material. vaccines from degradation but also improve permeation, uptake, and bioavailability of the drug. They further defined Bioavailability of CMs the parameters, such as size and distribution, of CMs that Most vaccines are administered by parenteral injection are important for improving drug bioavailability, reproduc- because the bioavailability of mucosally delivered vaccines ibility, and repeatability as well as steady release behavior. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress Producing equal sized CMs is very difficult, and the size Interaction with anions distribution would be too broad if the microspheres are pre- Ionotropic gelation pared by mechanical stirring or ultrasonication technique, The counterions that are used in the ionotropic gelation which are common methods for CM preparation. These method can be divided into two main categories: low could limit their vaccine delivery application. Firstly, the MW counterions (eg, pyrophosphate, tripolyphosphate, poor reproducibility of equal sized CMs may result in poor tetrapolyphosphate, octapolyphosphate, hexametaphosphate, repeatability on release behavior and efficacy among the octyl sulfate, lauryl sulfate, hexadecyl sulfate, and cetyl different batches. Secondly, the therapeutic efficacy can stearyl sulfate) and high MW counterions (eg, alginate, hardly be achieved with irregular sized CMs and a broad κ-carrageenan, and polyaldehydrocarbonic acid). Briefly, size distribution. Thirdly, a broad size distribution of CMs chitosan solution is added dropwise into magnetically stirred would result in poor bioavailability of the vaccine. Fourth, the aqueous counterions. The beads are removed from the solu- side effects of vaccine therapy would likely be increased. tion by filtration, washed with distilled water, and dried. Therefore, particle size is an important factor that should be CMs encapsulated with an atrophic rhinitis vaccine prepared taken into account in the application and pharmacodynamic by ionotropic gelation were nasally administered, which effect of vaccine-loaded CMs. Thus, it is important to prepare enhanced cytokine (tumor necrosis factor-α [TNF-α]) and CMs of uniform size with a narrow size distribution and nitric oxide production as an indication of immune stimulat- controlled release profile for their effective application in ing activity. mucosal vaccine delivery. Emulsification and ionotropic gelation Low off-target immunogenicity of CMs In the emulsification and ionotropic gelation method, an One of the major concerns of a vaccine carrier system is aqueous solution of chitosan is added to a nonaqueous the unwanted immunogenicity and pathogenicity caused continuous phase (isooctane and emulsifier) to form a water- by off-target reactions between the carrier itself and the in-oil emulsion. Sodium hydroxide solution is then added body’s immune system. This is the major disadvantage of at different intervals, leading to ionotropic gelation. The using bioengineered viruses or bacteria as delivery vehicles microspheres, thus formed, are removed by l fi tration, washed, for vaccines. Therefore, polymeric carriers have been and then dried. It has been suggested that the conventional investigated as a useful alternative for the efficient delivery emulsic fi ation and ionotropic gelation method for preparation of vaccines without unwanted immunological outcomes. of CMs provides irregular microparticles, whereas spheri- In this regard, chitosan can be considered a powerful cal microparticles with a diameter of about 10 µm can be polymer candidate because it has enormous potential for obtained when employing a modified process. In one modi - use as a vaccine carrier system that possesses low off-target fied process, gelatin is used, which allows the ionic crosslink - immunogenicity, suggesting that it will limit unwanted ing of chitosan/gelatin (water-in-oil emulsion) to take place off-target immune reactions with the body’s normal immune under coagulation conditions at a low temperature. Several function and not interfere with the actual vaccine-mediated other crosslinking agents have been used for surface modi- immune response which is to be loaded. Several reports have fication of chitosan/gelatin microspheres: the surface was also suggested that chitosan and its derivatives could be use- very smooth in sodium sulfate or sodium citrate crosslinked ful for drug delivery application without any significant off- chitosan/gelatin microspheres; however, large gaps were 47,48 target immunogenicity. Therefore, CMs (without vaccine observed in chitosan/tripolyphosphate microspheres. It has loaded) are expected to neither alter normal immunological been reported that the increase of stirring speed leads to a activity and biological function in the body nor interfere decrease in diameter and a narrower size distribution. with the vaccine efficacy by showing unwanted off-target immunogenicity. Complex coacervation Sodium alginate, sodium carboxymethyl cellulose, Preparation of CMs κ-carrageenan, and sodium polyacrylic acid can be used for Different methods have been studied and applied to pre- complex coacervation with chitosan to form microspheres pare CMs for the delivery of drugs and vaccines. Several after the interionic interaction between oppositely charged methods are discussed here in detail and are summarized polymers. For example, potassium chloride and calcium in Table 1. chloride were used to formulate the coacervate capsules of submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Table 1 Advantages and disadvantages of chitosan microspheres prepared by various methods Method Advantages Disadvantages References Interaction with anions Ionotropic gelation – These processes are simple and mild and have the following advantages: – Release of vaccine depends on various factors such as molecular 8,11,49 Emulsification and (a) use physical crosslinking by electrostatic interaction instead of weight, degree of deacetylation, and concentration of chitosan 50,51 ionotropic gelation chemical crosslinking; (b) reduce the possible toxic side effects of using and/or vaccine. Control of these factors by applying these 52,54 Complex coacervation various chemicals or reagents; (c) better control of degradation kinetics methods is sensitive to the preparation of chitosan microspheres Crosslinking with other chemicals Emulsion crosslinking – Easy to control particle size – Tedious process, uses harsh crosslinking agents 17,55,56 method – High drug loading efficiency – Crosslinking agent sometimes reacts with active agent – Controlled release with improved bioavailability – Complete removal of unreacted crosslinking agent is a challenge of this method Multiple emulsion method – Improves entrapment efficiency and loading content – Cannot avoid the use of organic solvent and crosslinking agent 12 – Better morphological characteristics – Improves production yield Thermal crosslinking method – Provides suitable particle size – Controlling the temperature is crucial because entrapment 57 efficiency and release of vaccine depends on a controlled temperature during the crosslinking process Crosslinking with a naturally – Naturally occurring agents are used as crosslinkers, thus less toxic – The physical, mechanical, and thermal stability of the 58–60 occurring agent – Smaller particle size, low crystallinity, and good sphericity microspheres prepared by this method are not well established; – Shows superior biocompatibility more investigation needed – Exhibits slow degradation rate compared to glutaraldehyde crosslinked chitosan microspheres Emulsion droplet coalescence – High loading efficiency – Particle size depends on the degree of deacetylation of chitosan. 61 method – Smaller particle size The decreased degree of deacetylation increases particle size which in turn decreases drug content Coacervation or precipitation – The process avoids the use of toxic organic solvents – Partially protects the loaded active agent from nuclease 63 method – Particle size and drug release can be controlled degradation Reverse micellar method – Smaller particle size and narrow size distribution – A tedious preparation process with many steps 64 – Thermodynamically stable particle size with suitable polydispersity index Sieving method – A simple method which is devoid of tedious processes – Irregular particle shape 65 – Can be easily scaled up Solvent evaporation method – Good entrapment efficiency and particle morphology – Controlling particle size depends on using agglomeration 66 preventing agent – Particle size decreased with the use of an increased amount of this agent Spray drying method – A popular method to prepare powder formulation – Control of size depends on size of nozzle, spray flow 67 – Good drug stability, good entrapment efficiency, and prolonged rate, pressure inlet air temperature drug release can be achieved – Entrapment efficiency depends on the molecular weight of chitosan, ie, chitosan with low molecular weight provides better entrapment efficiency Islam et al Dovepress chitosan–alginate and chitosan–κ-carrageenan, respectively, CMs had better biocompatibility and slower degradation rate 59,60 and the obtained capsules were hardened in the counterion than glutaraldehyde crosslinked CMs. The microspheres 52–54 solution before washing and drying. used as an injectable chitosan-based drug delivery system revealed low toxicity. Crosslinking methods Emulsion crosslinking method Emulsion droplet coalescence method Water insoluble reagents can be simply dispersed in chitosan Tokumitsu et al developed the emulsion droplet coalescence solution and entrapped by the emulsion crosslinking process. method for CM preparation, which implements the principle Glutaraldehyde, formaldehyde, and genipin have been widely of both emulsion crosslinking and precipitation. In this used as crosslinking agents for the preparation of CMs. In method, precipitation is usually induced by coalescence of the emulsion crosslinking method, chitosan solution is first chitosan droplets with sodium hydroxide. Briefly, a drug prepared by dissolving chitosan with acetic acid. This solu- containing stable emulsion solution of chitosan is prepared in tion is then added to liquid paraffin containing a surfactant, liquid paraffin oil. This emulsion is mixed with another s table forming a water-in-oil emulsion before the addition of a emulsion containing a chitosan aqueous solution of sodium crosslinking agent. The formed microspheres are filtered, hydroxide with high-speed stirring, which allows the drop- 17,55,56 washed with suitable solvent, and dried. lets of each emulsion to collide randomly and coalescently. This results in the precipitation of chitosan droplets with Multiple emulsion method small particle size. CMs loaded with gadopentetic acid were The multiple emulsion method is probably the best way to prepared using this method for gadolinium neutron capture increase the entrapment efficiency of the target molecule therapy. Gadopentetic acid interacts electrostatically with in CMs. In this method, a primary emulsion (oil-in-water) amino groups of chitosan since it is a bivalent anionic is first formed (nonaqueous solution containing the target compound. A range of nanosized particles and a high loading molecule in chitosan solution). This primary emulsion is of gadopentetic acid were obtained through this emulsion then added to an external oil phase to form multiple emul- droplet coalescence method compared to the conventional sions (oil-in-water-in-oil) followed by either the addition of emulsion crosslinking method. glutaraldehyde (as a crosslinking agent) or the evaporation of an organic solvent. CMs, loaded with hydrophobic reagents, Precipitation or coacervation method were found to have better morphological characteristics and Chitosan precipitates when it interacts with an alkaline yield when prepared by the multiple emulsion method. solution since it is not soluble in an alkaline pH medium. In this method, chitosan particles are prepared by dropping Thermal crosslinking method chitosan solution into an alkaline solution (eg, sodium In the thermal crosslinking technique, CMs are prepared with hydroxide, sodium hydroxide–ethanediamine, or sodium different thermal conditions in various steps. Orienti et al hydroxide–methanol) through a compressed air nozzle, reported CM preparation by the thermal crosslinking method which produces coacervate droplets. Particles are collected using citric acid, which served as crosslinking agent. Citric by precipitation or centrifugation before excessive washing acid was added to chitosan solution in acetic acid (2.5% with hot and cold water, respectively. The particle sizes weight/volume) and then cooled to 0°C before adding to can be controlled by varying the diameter of the compressed corn oil. After stirring for 2 minutes, the emulsion was then air nozzle together with the pressure. A crosslinking agent added dropwise to corn oil by maintaining the temperature can also be used to harden the particles, which would be at 120°C. Then, the crosslinking was performed under vigor- beneficial because of its slow release. Sodium sulfate was ous stirring (1000 rpm) for 40 minutes and the microspheres also used to prepare CMs using this precipitation technique. obtained were filtered, washed, dried, and sieved. Recombinant human interleukin-2-loaded CMs were pre- pared by a dropwise addition of sodium sulfate-containing Crosslinking with a naturally occurring agent recombinant human interleukin-2 solution in acidic chi- Genipin, a naturally occurring crosslinking agent, has also tosan solution. As a result, chitosan was precipitated and been used to prepare CMs by the spray drying method, recombinant human interleukin-2 was incorporated when which provides small particle size, low crystallinity, and CMs were formed. Of note, this method is devoid of any good sphericity. It was reported that genipin crosslinked crosslinking agent. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers Reversed micellar method Vaccine delivery through CMs Reverse micellar is the stable liquid mixture of oil, water, CMs have been examined for the mucosal delivery of and surfactants dissolved in organic solvents. To this mix- vaccines. A variety of chitosan-based carrier systems with ture, an aqueous solution of chitosan and the target molecule their functional properties for oral and nasal delivery is are added before the addition of a crosslinking agent such shown in Table 2. Here, the utility of CMs for oral and nasal as glutaraldehyde. Mitra et al described the preparation vaccination in vitro and in vivo are discussed. of doxorubicin–dextran conjugate-encapsulated chitosan Oral delivery nanoparticles. Oral delivery of vaccines has numerous advantages over conventional needle injection and is a well accepted route of Sieving method vaccination. However, most vaccines are still administered by Agnihotri and Aminabhavi developed a method to prepare injection due to the lack of a proper delivery system to reach the clozapine-loaded CMs. In this method, a thick jelly mass induction site and to enhance the effector responses. Although of chitosan was prepared in 4% acetic acid and crosslinked oral delivery is probably the preferred administrative route with glutaraldehyde. The crosslinked nonsticky jelly mass of vaccines, especially for children, it causes degradation of was passed through a sieve to get microparticles of a suitable the antigens in the gastrointestinal track and also shows inef- size, which were then washed with 0.1 N sodium hydroxide ficient targeting to the site of action when delivered in a naked to remove unreacted glutaraldehyde and dried overnight at form. Therefore, developing an effective delivery system 40°C. As a result, a high loading efficiency of clozapine has been considered the primary task in the oral vaccination (98.9%) was achieved. However, the particles were irregular e fi ld. To gain adequate immune responses after oral delivery, in shape with an average size of 543–698 µm. The irregular the vaccine should reach the M-cells of Peyer’s patches in shape and size of the particles is one of the major disadvan- the gut avoiding the acidic pH condition of the stomach and tages of this method, which could affect the bioavailability of enzymatic degradation. Even if the vaccine nearly reaches CMs in vivo. However, an in vitro and in vivo study demon- Peyer’s patches, the immune response is not always induced strated a controlled and sustained release of the drug. due to the inability of antigens to gain access to Peyer’s patches and because of inefc fi ient uptake at the induction site. Several Solvent evaporation method studies have shown that the uptake by M-cells was significantly The solvent evaporation method involves the formation of enhanced and degradation of protein and peptide vaccines in emulsion between a polymer solution and an immiscible the gastrointestinal track was prevented after the incorporation continuous phase – either aqueous (oil-in-water) or non- 59,68–71 of vaccine with CMs. Due to its nontoxicity and potent aqueous (water-in-oil). This can be done by using liquid antigen binding properties, chitosan has been considered a parafn fi /acetone. The target molecule dissolved in acetone is promising tool for oral vaccination. dispersed in chitosan solution and the mixture is emulsified Extensive research on CMs for mucosal vaccine delivery, in liquid paraffin while stirring. The microsphere suspen - in particular, the uptake of CMs in murine Peyer’s patches sion is filtered, washed, and dried. Magnesium stearate can in vitro and in vivo, was carried out by van der Lubben be added as an agglomeration preventing agent. It appears 59,68,70,71 et al. They prepared a human intestinal M-cell model that the average particle size decreases when the amount of by coculturing Caco-2 and Raji-cells and investigated the magnesium stearate used in the preparation is increased. uptake of CMs. No morphological changes in the mono- Spray drying method layer were observed and this model was used to examine Spray drying is one of the most widely investigated methods the in vitro uptake of CMs for oral vaccine delivery. They of preparing CMs in which chitosan solution is sprayed and found that CMs can be taken up by the epithelium of Peyer’s then air-dried followed by the addition of a crosslinking patches. It has been reported that the size of microparticles agent. He et al prepared CMs by spray drying multiple emul- should be ,10 µm for efficient uptake by M-cells and to sions (oil-in-water-in-oil or water-in-oil-in-water) to entrap reach the dome of Peyer’s patches. Indeed, CMs used in the cimetidine and famotidine into microspheres. The drug was study were much smaller than 10 µm and therefore suitable released in a sustained and controlled fashion compared to for M-cell uptake. Since chitosan is biodegradable, van der the other microspheres prepared by traditional spray drying Lubben et al further claimed that antigen was freed from CMs 67 68 or the oil-in-water emulsion method. after uptake by M-cells. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress fi submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Table 2 Chitosan-based carrier systems with functional properties for the delivery of (model) vaccines through oral and nasal routes Chitosan-based carrier type Delivery route (Model) vaccines Functional properties References Chitosan microparticles Oral Ovalbumin – Targets Peyer’s patches for M-cell uptake 59,68 Chitosan microparticles Oral Tetanus toxoid – Strong systemic and local immune responses 69 Chitosan microparticles Oral/ Diphtheria toxoid – Enhancement of both systemic and local immune responses 70 nasal Eudragit -coated chitosan Oral Ovalbumin – A controlled release profile of drug from the microspheres toward Peyer’s patches 72 microspheres – Induces proper immune stimulation Thiolated Eudragit-coated Oral Bovine serum albumin – Retains structural integrity of protein 4 chitosan microspheres – Improvement of mucoadhesiveness and residual time at the target site Chitosan microspheres (mixed Oral Hepatitis B surface antigen – Enhancement of antigen stability 73 with protease inhibitors – Strategic potential against chronic hepatitis B and permeation enhancer) Albumin–chitosan mixed matrix Oral Typhoid vi antigen – Induction of antigen-specific systemic and mucosal immune response 74 microspheres Chitosan microspheres Nasal Bordetella bronchiseptica – Shows suitable, but with some aggregation, physicochemical properties 49 dermonecrotoxin – Enhances immune stimulating activity in vitro and in vivo Pegylated chitosan microspheres Nasal B. bronchiseptica dermonecrotoxin – Improves stability and avoids aggregation of the microspheres 92 – Improvises immune stimulatory activity compared to chitosan microspheres alone Mannosylated chitosan Nasal B. bronchiseptica dermonecrotoxin – Specifically targets macrophages through the mannosylated moieties of mannose 91 microspheres receptor on the cell surface – Increases immune stimulatory activity in vitro and in vivo through specic targeting and activation of macrophages Chitosan microspheres Nasal N/A – No perceptible toxic effects 95,96 or chitosan solution alone – Increases bioadhesive properties Heat-labile toxin formulated Nasal LTK63 mutant of heat-labile – Induces high antigen-specific systemic and mucosal immune response 88,97 chitosan or N-trimethyl toxin (as adjuvant) chitosan microspheres Chitosan–DNA nanospheres Nasal DNA encoding Respiratory – Strong cell-mediated immune response 98 syncytial viral antigens Antigen-loaded chitosan/ Nasal Tetanus toxoid, diphtheria toxoid, – Stabilization of protein antigen by F127 105,106 Pluronic F127 microparticles and anthrax recombinant protective – Antigen stabilization strongly enhances the systemic and mucosal immune antigen response of chitosan/F127 than that of chitosan microparticles alone Dovepress Chitosan microspheres as vaccine carriers Therapeutic use of CMs for oral and nasal delivery protect chitosan from the acidic stomach. When this reaches has been examined. A diphtheria toxoid (DT) was used the intestine, the enteric layer dissolves at high pH and the to examine the enhancement of both systemic and local antigen-encapsulated chitosan core is exposed to enzymes. immune responses. Unloaded CMs, DT-loaded CMs, and In this state, chitosan can protect the encapsulated antigen DT in phosphate-buffered saline (PBS) were delivered into from enzymatic degradation and most importantly can lead mice by oral and nasal administration. DT associated with the antigen to reach the induction site of Peyer’s patches for alum was subcutaneously immunized in mice as a positive immune stimulation. For this, Hori et al developed Eudragit - control. A strong systemic and local immune response was coated CMs and evaluated ovalbumin as an oral immune found against DT in mice administered orally with different delivery system. The ovalbumin-loaded CMs prepared doses of DT-loaded CMs when compared to the mice fed by the emulsification-solvent evaporation method showed with DT in PBS. Furthermore, a dose-dependent anti-DT high ovalbumin content and an appropriate size for the immunoglobulin G (IgG) response in sera was found after efficient uptake by Peyer’s patches. A comparable systemic oral administration of DT-loaded CMs. On the other hand, the IgG response was found after the oral administration of systemic immune response (IgG) induced by DT-associated ovalbumin-loaded CMs in mice. Moreover, a higher intestinal CMs were ten times higher than that induced with DT in PBS mucosal IgA response was achieved using ovalbumin-loaded after nasal delivery. CMs by delivery of the microspheres toward Peyer’s patches, CMs were also examined after oral delivery of tetanus where they were subsequently uptaken by the M-cells and the 69 72 toxoid (TT) to induce systemic and local immune responses. entrapped ovalbumin was released in a controlled fashion. TT-loaded CMs were prepared by the ionic crosslinking In another study, Cho et al reported a mucoadhesive and pH- method using sodium tripolyphosphate. Unloaded CMs, sensitive thiolated Eudragit-coated CM, designed to enhance TT-loaded CMs, and naked TT in PBS were orally adminis- mucoadhesivity and bioavailability of the carrier at the target tered in mice, and TT absorbed on aluminum phosphate was site. They found strong mucoadhesive properties in vitro administered intramuscularly as a positive control. TT-loaded and in vivo, suggesting that Eudragit-coated CMs were a CMs enhanced a strong systemic and local immune response potential carrier for the oral delivery of vaccines. in a dose-dependent manner at 3 weeks after the oral delivery Recently, hepatitis B surface antigen-loaded CMs were of vaccine compared to TT in PBS. They observed that a formulated, characterized, and optimized in vitro and in vivo four-fold higher dose was needed for TT-loaded CMs to get for effective oral delivery of hepatitis B surface antigen a similar IgG response to the positive control. The study was against chronic hepatitis B. An emulsion solvent evapora- also carried out at different time points to understand the tion technique was applied to prepare CMs, with the addition kinetics of the immune response based on the level of IgG. It of protease inhibitors and permeation enhancers to overcome was found that the IgG response could be observed at day 14 the limitation of the enzymatic and permeation barrier. and was increased after boosting at day 22. At day 29, the IgG In vitro drug release, in vivo efficacy, and importantly the level was lower than at day 22; however, it still maintained effect of different storage conditions were studied to test a higher concentration than TT in PBS at all the time points the practicality of the system. An enhanced stability of the investigated. On the other hand, IgA levels were not sig- antigen was found when using the microspheres for a period nificantly different at day four; however, the levels were of 4 months at room temperature, suggesting a possible way significantly ( P , 0.01) higher in TT-loaded CMs than in TT to overcome the tedious and expensive requirement of cold in PBS at days eight, 14, and 22. These results suggest that chain storage in the vaccine industry. Importantly, the study the encapsulated vaccine in CMs enhanced the systemic as signifies a potential strategy for effective oral administration well as local immune responses compared to the nonencap- of hepatitis B surface antigen using the biodegradable CM sulated vaccine, rendering a safe and effective form of oral system. vaccination. Further studies on cellular immune responses Recently, Uddin et al developed an albumin–chitosan including memory effect of B-cells and T-cells will ensure mixed matrix microsphere (ACM)-l fi led capsule formulation the solid effectiveness of CMs for vaccine delivery. for oral administration of Typhoid Vi antigen (TVA) to At first glance, chitosan would not be considered sui t- demonstrate antigen-specic fi systemic and mucosal immune able for oral vaccination since it is a pH-sensitive polymer. responses. TVA-loaded ACMs were filled into hard gelatin It is soluble at acidic pH and becomes insoluble at about capsules with enteric coating. The physicochemical char- pH 6.5. It has been suggested that an enteric coating can acterization such as particle size, zeta potential, swelling, submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress and disintegration rates of the microspheres were favorable and a major virulence factor for atrophic rhinitis – a disease for oral delivery of the microencapsulated vaccine. In vivo that causes huge economic damage in the swine industry. studies showed that the oral delivery of TVA-loaded ACMs BBD-loaded CMs were prepared by the ionic gelation process had similar IgG and IgA responses with those of the par- using tripolyphosphate. The morphology of vaccine-loaded enteral vaccination group, suggesting that TVA-loaded microspheres was observed as aggregated shapes, whereas ACMs had the potential to induce antigen-specific immune unloaded microspheres were quite spherical. The average responses when delivered via oral administration. particle size of BBD-loaded CMs was 4.39 µm, which provides a condition for effective delivery of the vaccine Nasal delivery to nasal-associated lymphoid tissue for immune induction. Nasal administration of vaccines has been reported to The size of unloaded CMs was about 1.94 µm, indicating enhance bioavailability and improve efficacy. An effec- that CMs became enlarged after vaccine loading. The release tive humoral and cell-mediated immune response can be studies further demonstrated that when the MW of chitosan achieved through nasal delivery of vaccines when the decreased, more BBD was released. It was also found that appropriate delivery system is used as a carrier for particu- encapsulated BBD had greater release at higher pH than lower 75,76 late antigens. Nasal- associated lymphoid tissue, present pH. The secretion of TNF-α and nitric oxide from the murine at the nasal epithelium and containing immunocompetent macrophages treated with BBD-loaded CMs indicated that cells, would be an ideal target site for the nasal delivery of the cells stimulated with BBD-loaded CMs produced TNF-α 75,76 vaccines to induce an immune response. It has been sug- and nitric oxide in a time-dependent manner at a similar gested that nasal- associated lymphoid tissue epithelium has level to cells stimulated with BBD alone or lipopolysac- similar types of immune cells that are present in the M-cells charide. It is important to mention that BBD-loaded CMs of Peyer’s patches in gut-associated lymphoid tissue and induced a steadily increasing immune stimulating effect in is located just below the epithelial surface, which contains the macrophages, whereas it began to decrease at 80 hours macrophages, dendritic cells, lymphoid follicles (mostly poststimulation with lipopolysaccharide. B-cells), and intrafollicular areas (mostly T-cells) in a An in vivo study was carried out in mice that measured network. At these sites, particulate antigens are mainly taken IgG and IgA in sera, nasal wash, and saliva after intranasal up and/or transported across the cells by transcytosis without administration of BBD-loaded CMs. The IgA levels in nasal any extensive degradation. It has been well described that wash increased in a time- and dose-dependent manner after increased epithelial permeability influences the particulate intranasal administration of BBD-loaded CMs. However, such 77–84 antigen uptake across the epithelial mucosa. Importantly, immune response was not detected in saliva, suggesting that chitosan has the ability to increase membrane permeability CMs successfully delivered the vaccine to nasal-associated when used as a delivery system for nasal vaccination. lymphoid tissue after intranasal administration and induced However, antigen delivery through nasal administration a higher systemic and local immune response. Although in sometimes results in poor immune responses. Several factors vitro and in vivo results showed CMs as a potential carrier including limited diffusion of particulate antigens across the for nasal delivery, BBD-loaded CMs were found in aggre- mucosal barrier, rapid clearance of particulate drug or vac- gated shapes because of physical and storage instabilities. cine formulation from the mucosal surface, and enzymatic To overcome this instability problem, chitosan was degradation because of instability of the particulate carrier are modified by covalent conjugation with polyethylene glycol 86,87 92 associated with this. In order to overcome these problems, to form pegylated chitosan. The pegylated CMs (PCMs) chitosan might be one of the best options for nasal administra- were prepared through a similar ionic gelation process. The tion of vaccines due to its ability to increase the retention time average particle size of BBD-loaded PCMs was ,10 µm, when it binds to the mucosal membrane. Several reports their shape was spherical, and they were physically more also demonstrated that chitosan enhanced mucosal absorp- stable compared to BBD-loaded CMs. Due to better sta- tion of vaccines with adjuvant activity to improve mucosal bility, the vaccine was released from BBD-loaded PCMs 10,88,89 immunity after nasal administration. in a more steady fashion than in BBD-loaded CMs. The Cho and colleagues conducted extensive research on CMs study further showed that macrophages secreted TNF-α for intranasal delivery of vaccines to induce the immune and nitric oxide in a time-dependent manner after expo- 49,90–94 response in vitro and in vivo. They used Bordetella sure to BBD, BBD-loaded CMs, BBD-loaded PCMs, and bronchiseptica dermonecrotoxin (BBD), a causative agent lipopolysaccharide. However, a significantly higher TNF- α submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Dovepress Chitosan microspheres as vaccine carriers secretion was found in the cells treated with BBD-loaded used as a nasal vaccine delivery carrier without any harmful 95,96 PCMs than cells exposed to BBD-loaded CMs and BBD effects. To investigate this, the cilia beat frequency was alone. Moreover, TNF-α secretion increased in a sus- studied in guinea pigs after nasal administration of chitosan tained fashion in the cells exposed to BBD-loaded PCMs, solution for 28 days and found that none of the chitosan whereas it began to decline at 48 hours poststimulation induced the changes of cilia beat frequency, indicating a 92 95 with lipopolysaccharide. safety profile of chitosan for nasal delivery. They further To increase the target specificity, another study was investigated the bioadhesive properties of CMs via nasal carried out with mannosylated CMs (MCMs) with encap- administration using three different formulations: chitosan sulated BBD to target macrophage mannose receptors and solution, CMs, and starch microspheres, which was fol- increase immune stimulating activity. Colocalization of lowed by the examination of clearance properties in human BBD-loaded MCMs and the macrophage receptors was con- subjects. The clearance rate was 21 minutes for the control, firmed by confocal laser scanning microscope. The results 41 minutes for the chitosan solution, 68 minutes for the showed that macrophages exposed to BBD-loaded MCMs starch microspheres, and 84 minutes for the CMs. This secreted higher TNF-α and interleukin-6 than that of BBD- result indicates that CMs have better bioadhesive proper- loaded CMs and BBD alone. Furthermore, BBD-specific ties and are able to significantly reduce the drug clearance IgA response was found to be significantly higher in saliva rate and prolong the residence time of the delivered vaccine and serum after intranasal immunization with BBD-loaded in nasal mucosa, resulting in enhanced bioavailability and 91 96 MCMs in mice compared with BBD-loaded CMs, sug- efficacy. gesting that the MCMs extensively assisted in stimulating Several reports demonstrated the concomitant use macrophages for induction and enhancement of immune of CMs as a mucosal adjuvant and as a vaccine delivery activity. The representative scanning electron microscope system. A vaccine formulation with CMs and a nontoxic photographs of CMs and MCMs (BBD loaded and unloaded) LTK63 mutant of heat-labile toxin induced significantly are shown in Figure 2. higher IgG titers in sera and IgA in nasal washes after intra- Soane et al performed extensive research using differ- nasal delivery in mice. A modified N-trimethyl chitosan ent types of chitosan and concluded that chitosan could be microparticulate system also showed higher antigen-specific antibody responses in sera, nasal, and vaginal wash. Chitosan–DNA nanospheres with intranasal delivery exhib- ited significant responses of cytotoxic T-cell response and interferon-γ as well as antigen specific-IgG and IgA, render - ing a strong humoral and cell-mediated immune response. CMs were prepared with Pluronic F127 as an immuno- modulating and stabilizing agent to enhance the stability for controlled drug release and adjuvanticity. Pluronic, a triblock 5 µm 5 µm copolymer of polyethylene oxide and polypropylene oxide CMs BBD-CMs (polyethylene oxide-b-polypropylene oxide-b- polyethylene oxide) commonly known as poloxamer, has a variety of pharmaceutical applications and has become one of the most extensively investigated temperature-sensitive materials. F127 is water soluble and has a good drug release profile, which makes it a potent drug delivery carrier for a variety 100–104 of therapeutic and bioactive agents. When Westerink 5 µm 5 µm et al intranasally immunized antigen-loaded F127/CMs into MCMs BBD-MCMs mice, it signic fi antly increased systemic and mucosal immune Figure 2 Scanning electron microscope photographs of CMs, BBD-loaded CMs, responses compared to those of control groups, suggesting MCMs, and BBD-loaded MCMs (5000×). Notes: Bar represents 5 µm. Reprinted from Biomaterials, 29(12). Jiang HL, Kang ML, that the stabilization of protein antigens by F127 enhances the Quan JS, et al. The potential of mannosylated chitosan microspheres to target immune response of F127/CMs compared to chitosan alone. macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization, 1931–1939. Copyright 2008 with permission from Elsevier. This study demonstrated a nasal vaccine delivery strategy Abbreviations: BBD, Bordetella bronchiseptica dermonecrotoxin; CM, chitosan microsphere; MCM, mannosylated chitosan microsphere. for enhancement of the immune response via a synergistic submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Islam et al Dovepress effect of chitosan and F127. In another study, intraperitone- chitosan (eg, thiolated chitosan) might improve the stability ally and subcutaneously injected F127/cytosine–phosphate– and functionality of CMs. guanosine and F127/CM formulations signic fi antly enhanced antigen-specic fi systemic antibody responses compared to the Thiolated CMs as a modified antigens delivered with cytosine–phosphate–guanosine or and improved form of a chitosan- CMs alone, suggesting that F127 might have an adjuvant based mucosal vaccine carrier effect when used in combination with chitosan. Therefore, Thiolated polymers (ie, thiomers) have gained considerable application of a delivery system that combines adjuvants attention – especially for vaccine delivery – because they are with various modes of action is beneficial to maximizing one of the most promising polymers with multifunctional immune response. properties including strong mucoadhesivity, enhanced per- meation effects, protection ability, stability, and enhanced Limitations of CMs 109–114 bioavailability of drugs. Among various thiomer-based Besides the enormous advantages of CMs such as biode- carriers, thiolated CMs (TCMs) are highly popular because gradability, nontoxicity, permeation enhancing effects, and of their strong mucoadhesiveness and ability to control an ability to open the tight junction between epithelial cells and extend drug release profiles with improved permeation as described earlier, there are some limitations as well. 115–119 ability. TCMs can be prepared by immobilizing the Cho and colleagues performed several studies on CMs for thiol-bearing chain on the polymeric backbone of chitosan 49,90–94 vaccine delivery. They found that the vaccine-loaded (Figure 3). The strong mucoadhesivity of TCMs is obtained CMs self-aggregated at 2 weeks after preparation, although through the formation of disulfide bonds between the thiol it was effective in inducing immune responses including groups of TCMs and cysteine-rich subdomains of mucin cytokine expression in vitro and antigen-specic fi IgG and IgA glycoproteins at the mucosal surface (Figure 4). The per- 49,90 responses in vivo after nasal delivery. To make stable and meability through the mucosal surface can be enhanced by nonaggregated CMs, they used F127 to prepare F127/CMs which showed spherical morphology with no aggregation at an extended period of time after preparation. This was due CH OH CH OH CH OH 2 2 O O O to the hydrophilic polyethylene oxide chains of F127 that OH O O hindered the self-aggregation of CMs. F127/CMs showed OH OH OH OH much improved immune activity in vitro and in vivo and also NH NH NH exhibited potential protection against infection compared to 2 n 2 CMs alone. X Several other studies described the instability of CMs in SH acidic media, especially when prepared by the precipitation Thiolated chitosan method. CMs prepared by sodium sulfate precipitation were found to have poor acidic stability. This acidic instability was initiated by the addition of sodium sulfate to chitosan CH OH CH OH CH OH acetic acid solution which led to an ionic neutralization of O O O OH the positively charged amine groups of chitosan, providing O O OH OH OH poorly soluble chitosan derivatives. After the addition OH of acid (increasing proton concentration), the equilibrium NH NH NH 2 2 shifted to the solubilizing range for chitosan, thus dissolving the CMs. In another study, sulfadiazine-loaded chitosan NH beads were prepared using tripolyphosphate; however, it SH was found that the beads had poor mechanical strength. Collectively, there are some limitations of CMs that can Chitosan-N-acetyl-cysteine be overcome by modifying the CMs. For example, F127 Figure 3 Representative structure of thiolated chitosan: (A) general structure of is a good strategy to improve the stability and mechanical thiolated chitosan modified by an –SH group (X: linker) and (B) chitosan-N-acetyl- strength of CMs. Additionally, structural modifications of cysteine (modification of chitosan at the D-glucosamine unit by N-acetyl-cysteine). submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Mucin Mucin Mucin Mucin Mucin Mucin Dovepress Chitosan microspheres as vaccine carriers acid, thioglycolic acid, glutathione, and 2-iminothiolane are SH Mucin SH the aliphatic thiol-bearing ligands with functional carboxyl SH SH SH + Mucin TC TC + groups which form amide bonds with the amino groups of SH TC SH TC chitosan by carbodiimide to synthesize the thiomers of chi- SH TC Mucin TC SH SH + Vaccine 118,121–125 TC + tosan. CMs prepared by these thiomers exhibit strong TC SH Mucin TC + mucoadhesivity, biocompatibility, and enhanced permeability TC TC SH SH + TC + SH Mucin and absorption after oral and nasal administration. SH SH SH It is important to note that thiomers bearing free thiol SH Mucin groups are relatively unstable in solution because they are prone to oxidize at pH $ 5, leading to a self-crosslinking of the polymer. Different approaches have been attempted to delay oxidation and inhibit the self-crosslinking reaction. As an example of a next-generation thiomer, the aromatic thiol-bearing ligands are extraordinary candidates for delay- ing the oxidation process and protecting the thiol groups of the thiolated polymers. Recently, Bernkop-Schnurch et al TC TC + performed several studies using aromatic thiol-bearing ligands TC TC TC for the synthesis of S-protected thiolated chitosan and evalu- TC Vaccine + 109,127,128 TC ated their efficacy as mucosal drug delivery carriers. + TC TC TC TC To prepare the S-protected thiolated chitosan, the thiol-bearing TC + ligand was covalently attached to chitosan as the first step of modic fi ation. In the second step, the thiol group of thiolated chitosan was protected by the formation of disulfide bonds with aromatic thiol-bearing ligands. The S-protected thio- lated chitosan exhibited improved mucoadhesivity, enhanced permeation effect, inhibited efu fl x pump, bioavailability, and Thiolated chitosan SH Thiol group TC controlled release profile compared to the corresponding Thiolated chitosan 109,127,128 Mucin glycoprotein TCMs thiolated and unmodified polymers, demonstrating Mucin microspheres at mucus layer that TCMs prepared using S-protected thiolated chitosan are Disulfide bond a promising chitosan-based mucoadhesive polymer for the development of various mucosal vaccine delivery systems. Figure 4 Schematic representation of functional interaction between TCMs and mucin in mucosal vaccine delivery. Abbreviation: TCM, thiolated chitosan microsphere. Conclusion and future perspectives Among various investigated vaccine carriers, CMs hold using TCMs instead of unmodified CMs. Increased perme - enormous promise as a delivery vehicle for both oral and ability is achieved by opening the tight junction after the nasal administration. This review has discussed and evalu- inhibition of protein tyrosine phosphatase, a key enzyme ated various methods for preparation of CMs which could involved in the closing process of tight junction. Due to help to design more and better functionalized chitosan-based the formation of inter- and intramolecular disulfide bonds carrier systems. This study demonstrated that vaccine-loaded through TCMs, a compact three-dimensional network is CMs could be prepared with suitable and appropriate particle generated which allows controlled drug release and leads to sizes, which is a very important factor in the delivery of the high cohesivity. Moreover, TCMs exhibit a reversible opening vaccine to the induction site of mucosa-associated lymphoid of the tight junction, which leads to better permeation effects tissue for proper immune stimulation. Furthermore, both 115,117,120 than unmodified CMs. In the case of first-generation systemic and local immune responses can be induced in a thiomers, thiolated chitosan derivatives are prepared by con- dose- and time-dependent manner through vaccine-loaded jugating thiol-bearing aliphatic ligands to the amino groups of CMs. The nontoxic, highly bioavailable, mucoadhesive, chitosan. For example, N-acetyl-cysteine, 6-mecaptonicotinic and biodegradable nature of chitosan and its particulate submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2012:7 Dovepress Mucin Mucin Mucin Mucin Mucin Mucin Islam et al Dovepress 6. Eyles JE, Sharp GJ, Williamson ED, Spiers ID, Alpar HO. Intra nasal form is the main reason that it could become a successful administration of poly-lactic acid microsphere co-encapsulated Yersinia vaccine carrier in the near future. Furthermore, the much pestis subunits confers protection from pneumonic plague in the mouse. improved properties of modified CMs (eg, TCMs), such as Vaccine. 1998;16(7):698–707. 7. Janes KA, Calvo P, Alonso MJ. Polysaccharide colloidal par- increased mucoadhesivity, membrane permeability, stability, ticles as delivery systems for macromolecules. Adv Drug Deliv Rev. and controlled/extended release of the encapsulated vac- 2001;47(1):83–97. 8. Mi FL, Shyu SS, Chen CT, Schoung JY. Porous chitosan microsphere cine, show that they are a promising candidate for a potent for controlling the antigen release of Newcastle disease vaccine: vaccine carrier system. 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