century,it has only been over the last two decades that this polymer has received attention as a material for biomedical and drug delivery applications.The accumulated information about the physicochemical and biological properties of chitosan led to the recognition of this polymer as a promising material for drug delivery and,more speci?cally for the delivery of macromolecules[144–150].From a technical point of view,it is extremely important that chitosan is hydro-soluble and positively charged.These properties enable this polymer to interact with negatively charged polymers,macromole-cules,and even with certain polyanions upon contact in aqueous environment.These interactive forces and the resulting sol–gel transition stages have been exploited for nano-encapsulation pur-poses[5,151–153].On the other hand,chitosan has the special possibility of adhering to the mucosal surfaces within the body,a property leading to the attention to this polymer in mucosal drug delivery[148,150,154].The potential of chitosan for this speci?c application,has been further enforced by the demonstrated capacity of chitosan to open tight junctions between epithelial cells though well organized epithelia[155–160].The interesting biopharmaceutical characteristics of this polymer are accompanied by its well docu-mented biocompatibility and low toxicity[161–164].Many articles on the potential of chitosan for pharmaceutical applications have already been published[145,165,166].Therefore,our purpose is to focus on the speci?c features and applications of the chitosan-based nanoparticu-late systems prepared and characterized to date for delivery of macromolecular compounds such as peptides,proteins,antigens, oligonucleotides,and genes.
3.1.1.Chitosan-based nanoparticles with covalent crosslinks
The earliest works on chitosan-based nanostructures predomi-nantly involved chemical crosslinking within polymer chain.Watzke and Dieschbourg[167]formed chitosan/silica nanocomposites by reacting tetramethoxysilan with hydroxyl groups on the chitosan monomers.However,it was not attempted to associate pharmaceu-tically active agents to the prepared polymer network.Ohya et al.was the?rst to present data involving chitosan nanospheres for drug delivery applications[168].Using a water-in-oil(w/o)emulsion method followed by glutaraldehyde crosslinking of the chitosan amino groups,the group produced nanospheres loaded by5-?uorouracil(5-FU),an anticancer drug.Since5-FU derivatives in formulations also contained a terminal amine,glutaraldehyde addi-tion indiscriminately bound the active agent to the polymer as it did between chitosan chains,causing drug immobilization rather than encapsulation.These studies demonstrated the feasibility of synthe-sizing stable,reproducible nanosized chitosan particles which could entrap and deliver drugs[155].
3.1.2.Chitosan-based nanoparticles with ionic crosslinks
As mentioned,the cationic nature of chitosan has been conve-niently exploited for the development of particulate drug delivery systems.Aside from its complexation with negatively charged polymers,an interesting property of chitosan is its ability to gel upon contact with special polyanions,a process referred to as ‘ionotropic gelation’.This gelation process is due to the formation of inter and intra crosslinkages between/within polymer chains, mediated by the polyanions.More recently,chitosan NPs have been developed based on the ionotropic gelation of chitosan with tripolyphosphate(TPP),for drug encapsulation[169–174].This simple and straightforward technique involves the addition of an alkaline phase(pH=7–9)containing TPP into an acidic phase(pH=4–6) containing chitosan.NPs are formed immediately upon mixing of the two phases through inter and intra molecular linkages created between TPP phosphates and chitosan amino groups.
Insulin-loaded chitosan NPs have been prepared by mixing insulin with TPP solution and then adding the mixture to chitosan solution under constant stirring[175].Chitosan NPs thus obtained were within size range of300–400nm with a positive surface charge ranging from+54to+ing this method,insulin loading was optimized reaching the loading ef?ciency of up to55%.There are many ongoing investigations,which demonstrate the improved oral bioavailability of peptide and proteins upon undergoing this loading procedure.In these studies,it is claimed that the bioadhesion property of chitosan NPs further enhance the intestinal absorption of the drug. Pan et al.[176]prepared insulin-loaded chitosan NPs by ionotropic gelation of chitosan with TPP anions.The ability of chitosan NPs to enhance the intestinal absorption of insulin and the relative bioavail-ability of insulin was investigated by monitoring the plasma glucose level in alloxan-induced diabetic rats after oral administration of various doses of insulin-loaded chitosan NPs.The positively charged, stable chitosan NPs showed particle sizes within the range of250–400nm with insulin association ratio of up to80%.The in vitro release experiments indicated an initial burst phase which was pH-sensitive. The chitosan NPs enhanced the intestinal absorption of insulin to a greater extent than the aqueous solution of chitosan in vivo.After administration of21.1IU/kg insulin loaded in the chitosan NPs, hypoglycemia was prolonged over15h.The average bioavailability relative to the subcutaneous injection of free insulin solution was up to14.9%.
Xu et al.[177]have studied different formulations of chitosan NPs produced by the ionic gelation of TPP and chitosan.Transmission electronic microscopy(TEM)indicated particle diameters ranging between20and200nm with spherical shapes.
3.1.3.Chitosan-based nanoparticles prepared by desolvation method
The use of desolvating agents for the synthesis of chitosan particles originally emerged from the microencapsulation studies.Berthold et al.?rst proposed the use of sodium sulfate as a precipitating agent to form chitosan particles.Dropwise addition of sodium sulfate into a solution of chitosan and polysorbate80(used as a stabilizer for the suspension)under both stirring and ultrasonication,desolvated chitosan in a particulate form.Although the investigators called the resulting suspensions microspheres,the precipitated particles were at micro/nano interface(900±200nm).Drug encapsulation was not reported,but the group demonstrated that by virtue of the positive charge on the particle surface,they were able to absorb signi?cant amounts(up to30%loading)of the hydrophilic anionic corticosteroid, prednisolone sodium phosphate to the particle surface[178].A variation of this technique was later employed for the controlled release of antineoplastic proteoglycans for immunostimulation[179]. Following glutaraldehyde crosslinking of the nanoparticles,stable particles between600and700nm were obtained.Unfortunately,the necessity for glutaraldehyde forbids the application of this formula-tion toward the delivery of therapeutically active macromolecules. Chitosan-DNA NPs have been prepared using the complex coacerva-tion technique[165,180].At the amino-to-phosphate groups'ratio between3and8and the chitosan concentration of100mcg/ml,the particle size was optimized to100–250nm range with a narrow distribution.The chitosan-DNA NPs could partially protect the encapsulated plasmid DNA from nuclease degradation.
3.1.
4.Chitosan-based nanoparticles prepared by emulsion-droplet coalescence method
Emulsion-droplet coalescence method,introduced by Tokumitsu et al.[181],utilizes the principles of both emulsion crosslinking and precipitation.In this method,instead of crosslinking the stable droplets,precipitation is induced by allowing coalescence of chitosan droplets with NaOH droplets.A stable emulsion containing aqueous solution of chitosan along with the drug to be loaded is produced in liquid paraf?n.At the same time,another stable emulsion containing chitosan aqueous solution containing NaOH is produced in the same manner.When,?nally,both emulsions are mixed under high speed stirring,droplets of each emulsion would collide at random and
1642M.Hamidi et al./Advanced Drug Delivery Reviews60(2008)1638–1649