coalesce,thereby precipitating chitosan droplets to give small solid particles.In this study,Tokumitsu et al.prepared gadopentetic acid-loaded chitosan NPs by this method using100%deacetylated chitosan, with the mean particle size of452nm and drug loading ef?ciency of 45%.
3.1.5.Chitosan-based nanoparticles prepared by reverse micellar method
Reverse micelles are thermodynamically stable liquid mixtures of water,oil,and surfactant.Microscopically,they are homogenous and isotropic structures consisting of aqueous-in-oil droplets separated by surfactant-rich?lms.NPs prepared by conventional emulsion poly-merization methods are not only large(N200nm),but also have a broad size range.Preparation of ultra?ne polymeric NPs with narrow size distribution could be achieved by using reverse micellar medium [182].Aqueous core of the reverse micellar droplets can be used as a ‘nanoreactor’to prepare such particles.Since the size of this highly monodispersed and narrow size range reverse micellar droplets usually lies between1and10nm[183],they are among the promising NPs interested in drug delivery studies.Since micellar droplets are in Brownian motion in liquid medium,they undergo continuous coalescence followed by re-separation on a time scale that varies between milliseconds and microseconds[184].The size,polydisper-sity and thermodynamic stability of these droplets are maintained in the system by a rapid dynamic equilibrium.
In this method,the surfactant is dissolved in an organic solvent to prepare reverse micelles.To this,aqueous solutions of chitosan and drug are added gradually with constant vortexing to avoid any turbidity.The aqueous phase is regulated in such a way as to keep the entire mixture in an optically transparent microemulsion phase. Additional amount of water may be added to obtain NPs of large sizes. To this transparent solution,a crosslinking agent is added with constant stirring overnight.The maximum amount of drug that can be dissolved in reverse micelles varies from drug to drug and has to be determined by gradually increasing the amount of drug until the clear dispersion is transformed into a translucent solution.The organic solvent is,then,evaporated to obtain the micellar transparent drug mass.The remaining material is dispersed in water and then,by adding a suitable salt,the surfactant precipitates out.The mixture is, then,subjected to centrifugation.The supernatant solution is decanted,which contains the drug-loaded NPs.The aqueous disper-sion is immediately dialyzed through dialysis membrane for about1h and the liquid is lyophilized to drug powder.
Mitra et al.[185]have encapsulated doxorubicin-dextran con-jugate in chitosan NPs,using this method.
3.1.6.Chitosan-based nanoparticles prepared by self-assembly via chemical modi?cation
The self-assembly of chemically modi?ed chitosan into NPs has been investigated for the delivery of macromolecules[186–191]. Fractional conjugation of polyethylene glycol,PEG,via an amide linkage to soluble chitosan was shown to yield self-aggregation at basic pH[188].These aggregates could trap insulin following incubation in phosphate buffer saline(PBS),likely due to the electrostatic interactions between the unconjugated chitosan mono-mers and the anionic residues of the protein.Depending on the degree of PEGylation,aggregate sizes between5and150nm can be obtained. The degree of PEGylation also in?uences the release rate,as more extensively PEGylated aggregates release insulin more rapidly. However,it is dif?cult to draw conclusions based upon this data,as loading levels for the respective PEG formulations were not reported. An interesting approach leading to the formation of chitosan vesicles has been developed by Uchegbu et al.[189].They linked palmitic acid to modi?ed glycol chitosan chains,thus producing an amphiphilic polymer,which,upon mixing with cholesterol,formed nanovesicles approximately300–600nm in size.These vesicles demonstrated good biocompatibility,hemocompatibility,and stability in serum and bile salt.Moreover,the vesicles were able to encapsulate bleomycin,a chemotherapeutic agent.The loading process was performed via an ammonium sulfate gradient which drove the peptide into the vesicles.
Lee et al.[190]have investigated the effects of conjugating chitosan with deoxycholic acid in their attempts to design a new carrier for DNA delivery.Attachment of this hydrophobic moiety to soluble chitosan was found to have substantial effects on its aqueous stability, and the resulting amphiphilic macromolecule formed self-assemblies of self-aggregates upon sonication.The group has reported the ability of these self-aggregates to associate with DNA and transfect in vitro. Further work is currently underway aiming at gaining a better understanding of the arrangement of the deoxycholic microdomains imbedded within the chitosan aggregates[191].
3.2.Alginate-based hydrogel nanoparticles
Alginic acid is an anionic biopolymer consisting of linear chains of α-L-glucuronic acid andβ-D-mannuronic acid with properties such as a high degree of aqueous solubility,a tendency for gelation in proper condition with high porosity of the resulting gels,biocompatibility,and non-toxicity[192].Generally speaking,Sequential crosslinking and formation of polymeric networks,results in hydrogel structured drug delivery carriers such as micro-and nanoparticles upon the addition of counter-ions to alginate.Any possible cationic species can initiate the reaction sequence,but calcium chloride is favorably utilized by most researchers.The methods of preparation are usually determined with the aim to control the geli?cation phenomenon,which leads to desired size ranges depending on various factors including alginate concen-tration/viscosity,counter-ion concentration,the speed of adding counter-ion solution onto the alginate solution,etc.
In1993,Rajaonarivony et al.proposed a new drug carrier made up of sodium alginate[193].They represented alginate NPs with a wide range of particle sizes(250–850nm),formed within a sodium alginate solution following the addition of calcium chloride followed by poly-L-lysine.In this study,the concentrations of both polymer and counter-ion solutions were lower than those regularly used for gel formation. Additionally,with doxorubicin as the model drug,they reported that loading capacity could be reached at more than50mg of drug per 100mg of alginate.
Since the end of1990s until now,the number of studies involving alginate-based NPs is increasing[193–195],using the therapeutic agents such as insulin[196–198],antitubercular and antifungal drugs [199–202],and even it has shown promising remarks in the?eld of gene delivery[203].
While the size range of alginate NPs is greatly dependent on the order of addition of counter-ion to the alginate solution,some people claim bene?t from the addition of a polyelectrolyte complexation step in this procedure[197].Sarmento et al.prepared insulin-loaded NPs by alginate ionotropic pre-gelation followed by chitosan polyelecter-olyte complexation.In their effort,particles in nanometer size range were obtained under optimized condition with a loading capacity of 14.3%.In another study using dextran polysaccharide as the complex-ing agent,again,insulin was loaded in alginate-dextran nanospheres via nanoemulsion dispersion followed by triggered in situ gelation [198].The resulting particles ranged in size from267nm to2.76μm. Particles prepared demonstrated a unimodal size distribution and insulin encapsulation ef?ciency was reached to82.5%.
The failure of antitubercular chemotherapy is mainly attributed to the patient non-compliance to frequent long-term multidrug regi-mens.Interestingly,the application of modi?ed-release drug delivery systems provides a novel and sound prospective for the treatment of mycobacterial infections[200].In a study designed to evaluate the pharmacokinetic and tissue distribution of free and NP-encapsulated antitubercular drugs in different doses,alginate NPs containing isoniazid(INH),rifampin(RIF),pyrazinamide(PZA),and ethambutol (EMB)were orally administered to mice[199].The average size of NPs
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