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Hindawi Publishing Corporation International Journal of Carbohydrate Chemistry Volume 2011, Article ID 865704, 14 pages doi:10.


Review Article Chitosan-Grafted Copolymers and Chitosan-Ligand Conjugates as Matrices for Pulmonary Drug Delivery
Fernanda Andrade,1 Francisco Goycoolea,2 Diego A. Chiappetta,3, 4 Jos´ das Neves,1 e 3, 4 and Bruno Sarmento1, 5 Alejandro Sosnik,
1 2

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Rua An´bal Cunha 164, 4050-047 Porto, Portugal ı Institut f¨ r Biologie und Biotechnologie der Pflanzen, Westf¨ lische Wilhelms-Universt¨t M¨ nster, Hindenburgplatz 55, u a a u 48143 M¨ nster, Germany u 3 The Group of Biomaterials and Nanotechnology for Improved Medicines (BIONIMED), Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, 956 Jun´n Street, 6th Floor, 1113 Buenos Aires, Argentina ı 4 National Science Research Council (CONICET), C1033AAJ Buenos Aires, Argentina 5 CICS, Department of Pharmaceutical Sciences, Instituto Superior de Ciˆncias da Sa´ de-Norte, Rua Central de Gandra 1317, e u 4585-116 Gandra, Portugal Correspondence should be addressed to Bruno Sarmento, Received 28 April 2011; Accepted 21 July 2011 Academic Editor: Jianjun Li Copyright © 2011 Fernanda Andrade et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently, much attention has been given to pulmonary drug delivery by means of nanosized systems to treat both local and systemic diseases. Among the different materials used for the production of nanocarriers, chitosan enjoys high popularity due to its inherent characteristics such as biocompatibility, biodegradability, and mucoadhesion, among others. Through the modification of chitosan chemical structure, either by the addition of new chemical groups or by the functionalization with ligands, it is possible to obtain derivatives with advantageous and specific characteristics for pulmonary administration. In this paper, we discuss the advantages of using chitosan for nanotechnology-based pulmonary delivery of drugs and summarize the most recent and promising modifications performed to the chitosan molecule in order to improve its characteristics.

1. Introduction
Alongside the successful market launch of different products over the last decades, a continuous effort to formulate delivery systems for the pulmonary administration of a wide variety of drugs has been extensively described in the literature [1, 2]. The particular anatomical, physiological, and pathophysiological features of the respiratory tract pose enormous challenges that need to be overcome in order to obtain effective lung deposition, uniform distribution, in loco retention (main challenge is to circumvent mucocilliary clearance), and stability (particularly to enzymatic degradation) of therapeutic agents [1, 3]. Nevertheless, particular attention has been dedicated not only to drugs themselves but also to excipients required to improve the bioavailability of drugs administered pulmonarily.

In this context, excipients that could transiently enhance the absorption of drugs are on the spot. Chitosan, a polysaccharide with structural characteristics similar to glucosamines and obtained by the alkaline deacetylation of chitin, derived from the exoskeleton of crustaceans, is one of such appealing excipients. The safety and tolerability of chitosan are synergistic characteristics towards its application in drug delivery by different administration routes. Despite the natural properties, some drawbacks are associated with the poor solubility at physiologic pH and the passive targeting effect. Thus, chemical modifications of chitosan by conjugating various functional groups allow the control of the hydrophilicity and the solubility at neutral and basic pH and open new opportunities to expand the application of this biopolymer.

these effects were milder. which can also be regarded as potentially useful for the development of pulmonary drug delivery systems. this effect may be attributable not to the inherent toxicity of chitosan but most probably to the physical obstruction of the bronchioles due to higher viscosity of the chitosan formulation.3 mg/mL of nanoparticles were tested) to adenocarcinoma epithelial lung cell lines (Calu-3 and A549). Chitosan is a cationic mucoadhesive polymer. Moreover.. Also. antimicrobial [5] and antioxidant [6. The ability to establish ionic. either in the presence or absence of chitosan. Studies have also been conducted for derivatives of chitosan or chitosan-based formulations intended for pulmonary administration. stearic acid-g-chitosan oligosaccharide micelles presented an IC50 value of approximately 369 μg/mL [20]. Grenha et al. in particular macromolecules.e. General advantages include the wellestablished biocompatibility and biodegradability of chitosan [4]. Absorption enhancement was higher for higher MW isothiocyanate dextran. thiolation (i. The mucoadhesive properties of the native polymer can be further increased by chemical modification. immune stimulation may . attachment of side chains containing thiol groups) has been proved an interesting strategy for this last purpose [14]. glycol chitosan).and nanocarriers) that can be optimized in order to present optimal aerodynamic particle diameters for lung deposition and retention [8–11]. In the case of TMC. [15] reported on the enhanced mucoadhesion of nanoparticles composed of thioglycolic acid-glycol chitosan. [22] performed in vivo studies in rats by administering intratracheally chitosan and two brands of N-trimethylated chitosan (TMC). Toxicological data on chitosan has been extensively reviewed and is regarded to be favorable when considering its use as a pharmaceutical excipient [4]. 7] activities have been reported for different types of chitosan and derivatives.. the structural component of mucus fluids. However. the increment in pulmonary mucoadhesion observed by these researchers was correlated with the greater bioavailability of calcitonin when this peptide was associated with nanoparticles. International Journal of Carbohydrate Chemistry OH O HO O O HO O CH3 NH OH O O 2. presenting different degrees of substitution (20% and 60%). after intratracheal administration to rats. [19] showed that chitosan nanoparticles obtained by ionotropic gelation with tripolyphosphate and entrapped in mannitol microparticles presented reduced toxicity (concentrations of up to 1.e. In another study conducted in A549 cells. due to the transiently disruption of tight junctions (Figure 2) [16]. However. The processability of chitosan and several derivatives allow obtaining different types of systems (powders or wellstructured micro. the production of several proinflammatory cytokines has been observed for hydrophobically modified glycol-chitosan nanoparticles in vivo after intratracheal instillation in mice [24]. we revise some of the most recent and promising modifications performed to chitosan with special focus on its employment in the pulmonary delivery of drugs. which could be associated with lower viscosity when compared to chitosan. Histopathological analysis of lung tissue showed that chitosan elicited neutrophil infiltration and structural damage in the lung parenchyma. thus causing local asphyxiation. In one recent study. the presence of reactive amine groups grants chitosan the chemical versatility for modification and functionalization (Figure 1) [12]. providing evidence for the mechanism of intercellular tight junction disruption. Comparable inflammatory effects were also observed in rats after the intratracheal administration of chitosan microparticles [23]. chitosan and derivatives may also induce immune responses by lung cells/tissue. Results showed significantly higher permeation for all the investigated model drugs in the presence NH2 Figure 1: Chemical structure of chitosan. [17] performed in vivo experiments in guinea pigs by comparing the pulmonary absorption of different model drugs in solution (carboxyfluorescein and fluorescein isothiocyanate dextran with molecular weight (MW) varying from 4 to 70 kDa). Moreover. mucoadhesive properties of chitosan and derivatives may also be detrimental. as assessed by blood drug levels. Calu-3 cells exposed to chitosan microparticles were able to elicit the release of inflammatory cytokines (IL-2 and IL-8) [21]. since adhesion of delivery systems at the upper respiratory tract and airways can also occur. In an in vitro study. however. comprising N-acetyl-Dglucosamine (right) and D-glucosamine (left) units. Florea et al. Advantages of Chitosan for Pulmonary Delivery of Drugs Chitosan possesses different beneficial properties that make it an attractive option for designing adequate dosage forms and advanced drug delivery systems to be administered to or through the lung. For instance. of chitosan. hydrogen.2 In this paper. as compared to nanoparticles based on the nonthiolated polymer (i. in vitro cytotoxicity experiments were conducted for chitosan presenting different degrees of deacetylation and MW using human embryonic lung cells (L132 cells) [18]. More importantly. In order to confirm this effect in pulmonary drug delivery. evidences its potential for increasing lung retention of drug carriers comprising chitosan. and hydrophobic bonding with negatively charged chains of mucin [13]. thus limiting the amount of carrier that effectively reaches the deep lung. In particular. Results revealed that chitosan presented 50% inhibitory concentration (IC50 ) values higher than 1 mg/mL. Yamamoto et al. Chitosan is able to enhance absorption of drugs by the paracellular route. which is mainly absorbed by the paracellular route. Makhlof et al. Even if generally regarded as detrimental. In the particular case of pulmonary drug delivery. Formulating scientists should keep this phenomenon in mind when developing chitosan-based systems.

An increase in the degree of quaternization leads to increase of the permeation-enhancing effect of TMC [34].International Journal of Carbohydrate Chemistry 3 3 1 2 (a) (b) Figure 2: Effect of chitosan on the absorption of drugs by the paracellular route. TMC has CH3 CH3 Figure 3: Chemical structure of trimethyl chitosan (showing the modification at the D-glucosamine unit).5-. Chitosan and Chitosan-Grafting Copolymers for Nanoparticle-Based Pulmonary Drug Delivery Systems The first nanoparticles with drug delivery purposes began in the late 1960s [26]. 2. This capability as enhancer is due to opening the tight junctions between adjacent epithelial cells through interactions between the protonated (positively charged) amino groups on the C-2 position and the negatively charged sites on the cell membrane and/or in the tight junctions [37]. TMC-based nanoparticles have consistently shown their feasibility for mucosal immunization. respectively.. TMC is obtained by reductive methylation of chitosan using methyl iodide in the presence of a strong base (e. nanoparticles can be used therapeutically as carriers. 3. depending on the application field [27]. positive charges. at all degrees of quaternization [38]. and their transport across the nasal epithelium of rat was studied [42]. Florea et al. synergistic effects of the TLR9 ligand CpG in TMC particles have been reported after nasal vaccination [44]. A similar approach was assessed with the tetanus toxoid by nasal instillation in mice [41]. the bioavailability was enhanced by 2. and 3: represents chitosan. NaOH) at 60◦ C [33.g. Studies have addressed oral vaccination against Helicobacter pylori using urease [39]. or encapsulating the active substance (drug or biologically active material) or by adsorbing or attaching the active substance on the surface. 32]. observed that 20% and 60% N-TMC (TMC20 and TMC60) enhanced the permeation of octreotide in vitro by 21-.1. This soluble chitosan derivative has mucoadhesive properties and displays excellent absorptionenhancing properties. be a particularly interesting feature for vaccine development [25]. independently of the pH. TMC nanoparticles were loaded with FTIC ovalbumin. 1: represents the drug.9-fold. For pharmaceutical and medical applications. Trimethyl Chitosan (TMC). Trimethylated chitosan (TMC) is a partially quaternized chitosan derivative that is freely soluble in aqueous solutions over a wide range of pH as compared to other chitosan salt derivatives (Figure 3). 36]. and 3. in this section. Since this natural polymer offers remarkable advantages over other natural and synthetic polymeric carriers. O OH O HO H3 C N+ 3. although sometimes the term identifies particles in the 1 to 200 nm range. 2: represents the tight junction. Intranasal administration of the influenza antigen elicited local and systemic immune responses in mice [40]. either by dissolving. we will focus on chitosangrafting-based polymeric nanoparticles as drug carriers. entrapping. (a) Normal epithelium. In turn. even at neutral pH [35. Cell viability and histology studies showed that the TMCs are safer than chitosan and that Calu-3 cell monolayers are a valuable model for predicting the paracellular transport kinetics in . A recent study has also shown that the delivery of the model antigen ovalbumin (OVA) to the cervical lymph nodes in a nanoconjugated form with TMC (∼30 nm diameter) was twice more effective than the nasal administration of ovalbumin-containing TMC nanoparticles with size of one order of magnitude greater (∼300 nm diameter) [43]. 34].4-. 16-. (b) Transient disruption of tight junctions by chitosan with enhancement of drug absorption. Different nanoparticles have been developed for pulmonary administration of various drugs to treat diseases such as tuberculosis (TB) [28–30] and other pulmonary infections [31] and diseases [31. and 30-fold. Nanoparticles are solid colloidal particles made of macromolecular materials ranging in size from 1 to 1000 nm. Also.

apart from the reduction of the cytotoxicity. At room temperature the O-substitution is favored. O-. it is necessary to carry out studies to test the feasibility of these NPs as inhaled drug delivery systems.6%) leading to insulin loading values as high as 38.3. Like other derivatives. Shi et al. Control cells without any exposure to NPs and cells incubated with O-CMC NPs showed no fluorescence. In addition. Different substitutions patterns can be obtained according to the reaction temperature used (Figure 4). NSC was initially developed as a wound dressing material combined with collagen. they sustain that cationic polysaccharides are promising enhancers for peptide drug absorption with prospect for sustainedrelease formulations [22]. Conjugation of the antigen to TMC and TMC/OVA is therefore a viable strategy to increase the immunogenicity of subunit vaccines [45]. induced by loaded nanoparticles. that is. synthesized a new NSC derivative by means of microwave irradiation. Other studies have shown that. MCF-7. reported on the conjugation of an antigen. N-. Chitosan-g-PEG nanoparticles have been prepared by ionotropic gelation with TPP [57]. Hou et al. 3. PEGylation of TMC led to improved colloidal stability of polyplexes and significantly increased . the antitumor effect of NSCNP is achieved by necrosis and apoptosis induction in K562 cells. so that the particles can be “invisible” to phagocytic cells. and 48 h.O-. 10. the cell apoptosis rate was increased from 30% after 24 h to 62% at the end of 72 h. Conversely. Some authors have used this chitosan derivative to prepare anticancer-drug-loaded NPs. 3.4. different derivatives can be produced. Results of in vivo studies after intranasal administration to healthy rabbits showed that the plasma glucose levels fell sharply and remained at a low concentration for. 2-3 h and returned to baseline after 5 h. 14. PEGylated Chitosan. According to a cytomorphology study and the analysis of DNA fragments. Luo et al.6%. This system displayed a high association efficiency (>78.93. N. and 9. NSC is a chitosan derivative obtained by the incorporation of succinyl moieties into the N-terminal group of the glucosamine units (Figure 5) [52]. N-Succinyl-Chitosan (NSC). For example. prepared similar nanoparticles loaded with 5-fluorouracil (5-FU) [54]. cells incubated with curcuminO-CMC NPs displayed green fluorescence. Therefore. It is also recognized as an excellent cosmetic ingredient (Moistfine liquid. These nanoparticles were not tested for any specific route of administration.78. The uptake of TMCOVA conjugates by dendritic cells was similar to the uptake of TMC/OVA nanoparticles and over 5-fold greater when compared to a solution of OVA and TMC. 3. the O-CMC NPs without drug showed no cytotoxicity. In vitro studies showed that TPs were controlled released from nanoparticles in PBS at pH 7. administration in Sarcoma 180-bearing mice. and PC-3 cell lines.4. CMC is prepared by carboxymethylation of the hydroxyl and amine chitosan groups [47]. In another work. An interesting finding of this study was that TMCMCC composite nanoparticles obtained by electrostatic complexation of the two polymers with a positive surface charge exhibited higher immune responses when compared to chitosan. Grafting of hydrophilic polymers such as PEG onto chitosan is a well-known strategy to improve the solubility and biocompatibility of chitosan as well as to achieve lower recognition by the host immune system and increased blood circulation time (Figure 6) [56]. CMC-based nanoparticles of varying average size (40– 400 nm diameter) were developed for intranasal immunization [41]. INCI name Chitosan Succinamide). Yan et al. OVA. confirming the internalization of the particles [46]. NSC displays good water solubility in a broad pH range. Cellular uptake was analyzed by fluorescence microscopy. to TMC and the preparation of nanoparticles for subunit vaccination. Li et al. Taking into account these results.78 υg/mL at 12. TMC. For example. respectively. injection (close to the tumor) in mice bearing S180 sarcoma tumor [53]. prepared nanoparticles with oleoyl-carboxymethyl chitosan encapsulating rifampicin as drug delivery systems International Journal of Carbohydrate Chemistry [51]. CMC is prepared by adding a carboxymethyl group in the structure of chitosan. being found small quantities in kidney and spleen [54]. Due to the advantages that PEG confers to chitosan. evaluated antitumor effects of NSC nanoparticles (NSCNPs) without drugs in K562 cells [55]. chitosan-g-PEG copolymer has been prepared and utilized to develop various types of nanocarriers for transmucosal drug delivery. They evaluated biodistribution and tumor targeting after i. 36. But their pulmonary administration could be a possibility to treat tuberculosis.26.v. used different kinds of CMC with various molecular weights and degrees of substitution to prepare nanoparticles through ionotropic gelification with calcium ions. These results showed the feasibility of CMC nanoparticles to entrap doxorubicin and the potential to deliver it following a controlled profile [49]. Slutter et al. and MCC nanoparticles. In L929.c. while at higher temperature the Nsubstitution is the preferred pathway. This modification increases its solubility in neutral and basic solutions without affecting other important characteristics [46]. or N. CMC nanoparticles were prepared as carriers for some anticancer drugs. Taking into account reaction conditions and reagents.N-dicarboxymethyl chitosan [48].4 the airway epithelia. and it is considered biocompatible both in vitro and in vivo. Carboxymethyl Chitosan (CMC). These PEG chains create a barrier layer to prevent the adhesion of opsonins present in the blood. tea polyphenols (TPs) were loaded into carboxymethyl chitosan and chitosan hydrochloride [50]. whereas curcumin and curcumin-O-CMC NPs resulted in considerable cell death. prepared curcumin-loaded O-CMC nanoparticles (curcumin-O-CMC Nps) as a novel carrier in cancer drug delivery applications. at most. 24.2. The results revealed that NSCNP could inhibit the proliferation of K562 with an IC50 of 37. Anhitha et al. The 5-FU-loaded NPs were biodistributed mainly in the tumor and liver. They entrapped successfully hydroxycamptothecin (HCPT) into the NSC nanoparticles and observed tumor targeting and significant suppression of tumor growth after s.

Thiolated Chitosan. it was documented that thiolated chitosan has strong mucoadhesive properties ascribed to the formation of disulfide bonds with cysteine-rich domains of mucus glycoproteins. . up to 10-fold. lactobionic acid (LA) in chitosan [60]. 3. Insulin-loaded nanoparticles prepared with chitosan-Nacetyl-L-cysteine were found to improve the systemic absorption of insulin after nasal administration [62]. L929. Thiol modifications to chitosan and nanoparticles derived from it have been aimed to localize a drug delivery system at a given target site (Figure 7). Mao et al. (3) O-CMC. Recently. with higher depositions in kidney and liver [59]. For this. These improvements resulted in a significant. and MeWo cells. They conjugated PEG on the nanoparticles and observed that the clearance of the PEGylated nanoparticles in male AKR/J mice (6–8 weeks) following intravenous administration was slower than that of unmodified nanoparticles at 15 min. (2) N. Although the exact mechanism for the enhanced effectiveness of this Figure 6: PEG-g-chitosan as obtained by the two most commonly used synthetic routes: (1) reaction with an active ester derivative and (2) reductive amination. They observed that the system with ligand chitosan-(O-MPEG)-(N-LA) showed better transfection efficiency (45.International Journal of Carbohydrate Chemistry OH O O HO HO O O (1) O OH O O O HO NH2 O HO HO O (3) (4) (2) O OH O O NH O NH O HO HO N OH OH O 5 Figure 4: Chemical structure of different types of carboxymethyl chitosan (CMC): (1) N-CMC. prepared chitosan-DNA nanoparticles using a complex coacervation process [59].3%) than ligandfree chitosan-(O-MPEG) (19. OH O HO O NH OH O O Figure 5: Chemical structure of N-succinyl-chitosan (showing the modification at the D-glucosamine unit).8%). Other authors developed a functional nanoparticulate carrier for DNA transfection in asialoglycoprotein receptor overexpressed in HepG2 cells. leading to an improvement in mucoadhesion of up to 140-fold when compared to unmodified chitosan [61].OCMC (showing the modification at the D-glucosamine unit). and (3) N. X: linker. increase of transfection efficiency in NIH/3T3. OH O HO O O MeO O n OH O HO O MeO O n (2) O NH X O NH O (1) cellular uptake compared to unmodified TMC [58].N-CMC. they grafted a methoxy PEG (MPEG) and a receptor ligand.5.

with emphasis on poorly soluble anticancer drugs [69–73]. The concentration and the temperature above which the monomers turn in micelles are called critical micelle concentration (CMC) and critical micellization temperature (CMT). Chitosan-Based Polymeric Micelles for Pulmonary Drug Delivery Micelles are spherical nanosized colloidal dispersions with a hydrophobic core and hydrophilic shell. Recently. 67]. It is also possible to promote the targeting of drugs to specific organs and tissues using stimuli-responsive micelles [77–79] or modulating the micellar surface with active-targeting ligands [77. generally di. In this section. for pulmonary administration of various drugs to treat different diseases such as cancer [82]. much attention has been given to polymeric micelles formed by lipid polymer or block copolymers. Water-soluble drugs are adsorbed on the micelle surface. Furthermore. Chitosan-Based Micelles. The same research group developed SACSO micelles for the delivery of drugs like paclitaxel [89]. 77]. and pulmonary infections [84–86]. Two forces are involved in the micelle formation. and drugs with intermediate polarity are distributed along the amphiphilic molecules [66]. polymeric micelles can be a useful strategy to solve the problem of drug resistance [81]. By sharing some structural and functional features with natural transport systems. making these micelles a promising gene delivery system in the treatment of pulmonary diseases. polymeric micelles allow the encapsulation of drugs with different polarities. The self-assembly of amphiphilic molecules in water is driven by a gain in entropy of the solvent molecules and a decrease of free energy in the system as the hydrophobic components withdraw from the aqueous media [64]. as drug delivery systems. an attractive force that leads to the association of molecules and a repulsive force that prevents unlimited growth of the micelles [65]. Micelles can be used as drug delivery systems. Recently. micelles are poorly recognized by the reticuloendothelial system (RES) and present long circulation times in bloodstream with enhanced permeability and retention effects (EPR effect) at solid tumor sites or another areas with leaky vasculature such infarcts. They have been also studied as carriers of genetic material to treat cystic fibrosis [87].1. chitosan can be complexed with negatively charged DNA and be used as nonviral vector for gene therapy. system remains unclear. making them more physically stable. composed by am-phiphilic molecules which self-assembly under certain concentration and temperature conditions (Figure 8). Figure 7: Thiolated chitosan: (1) general structure of thiolated chitosan as modified by an –SH group and (2) chitosan-N-acetylcysteine (showing the modification at the D-glucosamine unit). and inflammations. for example. 91]. Due to its size. 4. and proteins [92]. inflammation conditions [83]. micelles have the ability to penetrate tissues. showing also a high encapsulation efficiency and possibility of sterilization by filtration [63. infections. The SA-CSO/DNA micelles efficiently protected the condensed DNA from enzymatic degradation by DNAse I and presented lower cytotoxicity and comparable transfection efficiency in A549 cells compared to Lipofectamine 2000 [88]. Due to its cationic nature. hence. it was speculated that the mucoadhesive and permeation-enhancing properties based on disulfide bond “anchorage” increase the accessibility of insulin molecules to the epithelial membrane and. 4. virus and lipoproteins. SA-CSO micelles were developed by another . 80]. Hu and coworkers synthesized stearic acid. The encapsulation of doxorubicin in SA-CSO micelles resulted in higher uptake and accumulation by A549 cells and a decrease in the IC50 [20]. polymeric micelles present a lower CMC value. facilitates the absorption of the protein across the nasal epithelium. Compared to micelles prepared from conventional detergents. for passive targeting [76. especially for poorly water-soluble drugs that are incorporated into the micelle core.and triblock copolymers. doxorubicin [90. Like liposomes. both in vitro and in vivo. However. they are more stable than liposomes. X: linker. generally lower than 200 nm. due to its general small size. 66. They have also been studied as carriers of genetic material [74] and diagnostic agents [75]. even under dilutions to final concentration below the CMC [68]. and hydrophilic surface.(SA-) grafted chitosan oligosaccharide (CSO) in order to produce polymeric micelles to delivery pDNA (pEGFP-C1) [88]. Many studies have been published in the last years regarding the use of polymeric micelles as solubilization agents and bioavailability enhancers of different drugs. in the last years. respectively [63]. Different polymeric micelles have been developed.6 OH O O HO X SH NH O (1) (2) NH O HO O SH OH O International Journal of Carbohydrate Chemistry CMC NH CMT Figure 8: Formation of micelles by self-assembly of polymer monomers above critical micelle concentration (CMC) and critical micellization temperature (CMT). we will focus on chitosanbased polymeric micelles as drug carriers.

the micelle size and drug-loading content increased. their application has been centered particularly in noninvasive routes of administration including transmucosal administration of proteins [121–125] and genetic material [126–130]. The present systems were not tested for any specific route of administration. its mucoadhesiveness [113. They also synthesized N-alkyl-N-trimethyl chitosan derivatives to deliver 10-hydroxycamptothecin. but significantly reduced toxicity and improved bioavailability [103]. resp. Another research group studied the feasibility of N-succinyl-N-octyl chitosan as delivery system of doxorubicin [105].(PLA-) chitosan copolymers with different molar ratios were developed and characterized. with reference to specific systems. The micelles possessed positive charges with mean diameters between 100 and 250 nm and were efficiently nebulized using an Air-jet nebulizer presenting up to 52% of fine particle fraction.and nanoscopic scales. mannose) or specific ligands such as folate. Nanocarrier systems of this kind with improved biological and biopharmaceutical performance have been the subject of active research in the past decade or so. and capacity to promote the absorption of poorly absorbable macromolecules across epithelial barriers by transient widening of cell tight junctions thus modifying the parallel transport [117–120] have been exploited in the development of nanocarrier systems for transmucosal delivery. as discussed. 131]. Conjugation of Sugar Ligands. 105]. Galactose groups were chemically bound to . Liu and coworkers developed an triblock copolymer consisting of poly(ε-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCLb-COS-b-PEG) for delivery of drugs. Polylactide. thiolated derivatives. As a consequence. Rifampicin was used as lipophilic drug model to be encapsulated. galactose. using chitosanpoly(ε-caprolactone)-poly(ethylene glycol) to encapsulate paclitaxel and rutin with glutaraldehyde after crosslinking [97]. Particularly. and presented similar antitumor efficacy as Taxol. a phenomenon intimately related to its aggregation state [95]. it is also possible to produce chitosan derivatives with amphiphilic characteristics that may self-assemble in aqueous environment and form polymeric micelles [99–101]. 7 Their pulmonary administration could be a possibility to treat locally lung cancer.. Genipin after crosslinking did not affect the macroscopic characteristics of the micelles but delayed the in vitro release of doxorubicin from the micellar reservoir [96]. The capacity of chitosan to undergo multiple chemical modifications has been exploited to increase the active targeting of chitosan-based nanocarriers particularly for protein and genetic material delivery towards specific cells [126. After encapsulation into micelles. 114]. Between the different derivatives. using doxorubicin as model drug [96]. making them a suitable choice for pulmonary delivery of AmB [93]. 5.1. biocompatibility [115. AmB presents the same antifungal activity of Fungizone but lower toxicity [93]. chitosan is an ideal natural polymer for the design and development of drug delivery systems structured at micro. avoids the systemic side effects and improves the bioavailability of the drug [94]. the N-lauryl-carboxymethyl-chitosan micelles developed by Miwa et al. [107]. The obtained polymer presents the capacity to form micelles with encapsulation efficiency of doxorubicin close to 50%. however. present in some patients receiving immune suppressive treatments. Examples are the N-mPEG-N-octylO-sulfate chitosan micelles produced by Qu and co-workers [106]. N-octyl-O-sulfate chitosan presented the best results in terms of solubilization of paclitaxel [102]. 112]. In the field of block copolymers. Chitosan-based polymeric micelles have also been developed as drug delivery systems for other routes of administration than pulmonary. As PLA molar ratio increased. with emphasis on intravenous administration of paclitaxel. Strategies to improve the targeting potential of chitosan have addressed its functionalization with sugar moieties. grafted PEG) to conjugation with biologically active ligands such as carbohydrates (e. 110] or the methoxy poly(ethylene glycol)grafted chitosan micelles for delivery of methotrexate to treat colon carcinoma [111. for the pulmonary administration of amphotericin B (AmB) [93].) [104]. and the rifampicin release rate decreased [98]. stability. they present antitumor efficacy against lung cancer cell lines such as Lewis lung cancer cells [103] and A549 cells [103.g. studies are required to access its feasibility as inhaled drug delivery systems. Zhang and co-workers synthesized different chitosan derivatives composed by long-chain alkyl groups as hydrophobic moieties and sulfated groups as hydrophilic moieties to delivery of paclitaxel [102]. or KNOB viral protein. for intercellular antitumor drug delivery to drug resistance tumor cells [109. Similar results were obtained by Chen et al. Other chitosan-based micelles have been developed such as the doxorubicin-loaded linoleic acidgrafted chitosan oligosaccharide micelles produced by Du et al. release behavior. in the following sections. The best results in terms of encapsulation efficiency. transferrin. and pharmacokinetic properties were obtained with Noctyl-N-trimethyl chitosan (degree of octyl and trimethyl substitution is 8% and 54%. 5.International Journal of Carbohydrate Chemistry research group. Chitosan-Ligand Conjugates for Nanoparticle-Based Active Target Drug Delivery Systems As discussed in previous sections. Wu and co-workers also synthesized chitosan-based copolymers for drug delivery [98]. 116].. Paclitaxel-loaded micelles prepared with N-octyl-O-sulfate chitosan had high drug-loading capacity and encapsulation efficiency.g. mostly with D-galactose and D-mannose. Although these systems have been tested for intravenous administration. The modifications have comprised from derivatization with small functional groups or substructures (e. were shown to be safe for intravenous injection. The local administration of AmB to treat invasive pulmonary fungal infections. and the N-octyl-N-(2-carboxylcyclohexamethenyl) chitosan micelles produced by Liu and co-workers [108]. Besides the creation of lipid-chitosan and polymer-chitosan conjugates. A recognized feature of chitosan-based nanostructured systems is their capacity to protect sensitive therapeutic macromolecules against degradation and their ability to overcome mucosal barriers.

Tf decoration of chitosan-based DNA-loaded nanoparticles has been found to enhance the transport across polarized monolayer Caco-2 cells known to have abundant Tf receptors on their surface [159] and extensively used as a model of normal intestinal epithelium transport [161]. such as transferrin and viral KNOB. the high affinity of folate to bind its receptor (1 nM) [137] and folate small size allows its use for specific cell targeting. These systems have been developed with a view to achieve targeting effect in the delivery of cytostatic drugs to tumor 5-fold. this system allowed for an increase in IL-1Ra expression combined with a diminution of cytotoxicity in vitro and reinforced protection against inflammation and abnormal bone metabolism in vivo. HepG-2 [153]. This system was found to enhance the transfection of HepG2 cells having ASGRr. The transferrin receptor (TfR) is found in many mammalian cells. which then complexed with pcDNA. It was demonstrated that Tf conjugation could enhance the transport of nanoparticles through Caco-2 alone and Caco-2-PPL cocultures by 3. To this end. Conjugation of Folate. This same system was tested in vivo by a different group. or antiarthritis therapies and also for diagnostic and imaging purposes. Using these nanoparticles in the delivery of a plasmid encoding IL-12 resulted in enhanced IL-12 gene transfer efficiency. It was found that this system has the potential to be a vector for targeting to Kupffer cells in vivo. International Journal of Carbohydrate Chemistry The evidence from most of these studies is consistent to indicate that the folic acid modification promotes the uptake of nanoparticles by FR-positive tumor cell lines most likely via receptor-mediated endocytosis but has little impact on other cells without FR [155]. The results led to suggest that uptake in the lymphoid follicles of the duodenum could play a more significant role compared to Peyer’s patches [157]. Caco-2 [143]. Other chitosan glycoconjugates have incorporated lactose to develop gene nanocarriers. GCP-DNA complexes were found to be stable due to hydrophobic shielding by PEG and increased the stability against DNAse degradation. Folic acid (FA) is appealing as a ligand for targeting cell membrane and allowing nanoparticle endocytosis via the folate receptor (FR) for higher transfection yields. Glycoconjugated chitosan was designed for ASGRr-directed delivery to liver parenchymal cells. which specifically recognizes the galactose ligands on modified chitosan [132].2. TEM evidence was consistent with the proposal that the nanocomplexes were internalized by HeLa cells and located inside endocytotic vesicles and endosome-like compartments [133]. Hence. This modification increased the carrier uptake and transfection efficiencies in various in vitro assays as well as in mouse lung tissue as recently reviewed elsewhere [136]. the majority of in vitro studies have been conducted in various types of cell lines well known to overexpress the human folate receptors (FRa and FLRb). the ability of FA to bind its receptor to allow endocytosis is not altered by covalent conjugation of small molecules [138]. as a strategy to achieve active targeting and thus high transfection efficiency [59. folate conjugation to the surface of chitosan and chitosan-derivatives-based nanoparticles has been one of the actively studied strategies to vectorize drugs over the past few years [140–149]. HeLa cells were effectively transfected by this nanocarrier system. In turn. Importantly. suppressed tumour growth and angiogenesis in the carcinoma BALB/c mouse model [134]. Compared to unmodified chitosan or naked DNA. 157]. 154] cells. The system was found efficient to transfect liver cells expressing asialoglycoprotein receptor (ASGRr). the Tf-decorated nanoparticles did not show significant enhancement of the transfection of HeLa cells [160]. Transferrin (Tf) or antibodies against TfR were conjugated to oligonucleotides or polycations. chitosan has been functionalized with mannose. Moreover. Two different synthetic approaches were tested to couple Tf to the surface of chitosan nanoparticles. KB [151. Galactosylated chitosan-graft-PEG (GCP) was developed for the same purpose. but neither HepG2 nor BNL CL2 cells. However. NPs made out of folategrafted chitosan were produced to transfect interleukin-1 receptor antagonist (IL-1Ra) in synovial mononuclear cells and CD14+ cells via the targeting of the folate receptor-b [156]. achieving a conjugation in both cases about 33–43% (transferrin to chitosan moL%) [59]. efforts have been made to target mannose receptors on dendritic cells residing in tumors. Proteins. B16F1 [150]. In turn. responsible for iron import to cells. A trisaccharide branch was attached onto chitosan chain in order to target lectins on the cell surface in lung tissues [135]. An interesting contribution of this study was to address the behavior of a coculture of polarized Caco-2 cells infiltrated with lymphocytes that induces the differentiation of M-type phenotype. In this study HEK293 cells were transfected using luciferase reporter gene. hence. genetic material. have been conjugated at the surface of chitosan-based nanoparticles intended for DNA delivery. and SKOV3 [141. HT29 [143]. thus indicating that galactosylated chitosan is an effective hepatocyte-targeted gene carrier [132]. such as HeLa [145]. a model much closer to the real intestinal epithelium. One drawback of these systems is that they only proved to be ineffective in the presence of added serum medium. 5. A second related strategy has been to decorate the surface of DNA-loaded chitosan-based nanoparticles with KNOB protein so as to enhance the uptake and overcome one of the major rate-limiting steps for transfection mediated by . and it is known to enhance the transcytosis of viral vector [158.8 chitosan aiming to achieve liver target delivery.3. while dextran was grafted to enhance the stability of the complex in aqueous media. Results of transfection studies showed that folate-chitosan-based nanoparticles enhanced the reporter gene expression against a cell line overexpressing FR (SKOV3 cells) compared to an FR-deficient cell line (A549 cells) and did not induce obvious cytotoxicity against HEK 293 cells [154]. Conjugation of Protein Ligands. 152]. 5. and the nonepithelial isoform of FR (FRb) is expressed on activated synovial macrophages present in large numbers in arthritic joints [139]. Folate receptor (FR) is over-expressed on many human cancer cell surfaces. Tf-conjugated nanoparticles invariably showed threefold greater transfection efficiency than unmodified nanoparticles. 159].

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