Main characteristics of nanoparticles based on quaternized chitosans
1. Introduction
A tremendous effort has been and is currently being devoted to the research in the field of pharmaceutical nanotechnology. Several peculiar properties of gelled polymeric nanosize (<1μm) particulate systems have been reported, among which the ability to encapsulate either small molecular weight or macromolecular active principles in mild conditions and protect them from degradation by the harsh pH conditions or enzymes they may encounter in the organism, promote transport of actives across mucosal barriers, undergo internalization by cells thereby carrying actives into them. Chitosan, a copolymer of glucosamine and
In the following sections the nanoparticles obtained from chitosan derivatives will be surveyed in respect to preparation procedures, interactions with cells and tissues, factors influencing biological properties, pharmaceutical applications.
2. Preparation procedures
2.1. Ionotropic gelation
Ionotropic gelation, that is by far the most used technique for preparation of nanoparticles from chitosan derivatives, was first reported in 1997 by Calvo et al. [13]. The basic concept is that a polycationic polymer in aqueous solution passes, in appropriate conditions, from sol to dispersed gel following electrostatic crosslinking with an adequate anionic substance. This technique has been used with several quaternized chitosans carrying fixed, pH-independent positive charges, the most known of which is
The nanoparticles prepared by ionotropic gelation of quaternized chitosans with TPP were generally 200-300 nm in size, i.e., smaller than those obtained by the same method starting from plain chitosan which, by the way, showed lesser stability and tended to re-dissolve after some time from formation. The zeta potential was always positive, in the 10-20 mV range. The solution of the chitosan derivative into which the TPP solution was dripped would often contain a surfactant, usually Tween 80, to hinder nanoparticle aggregation and facilitate their re-dispersion after centrifugation. In fact, centrifugation was necessary to clear the particles of non-encapsulated drug.
The technique under discussion has also been used to prepare nanoparticles from thiolated derivatives of chitosan [20-23].
These polymers have shown mucoadhesive properties due to the ability of their thiol groups to form covalent disulfide bridges by reacting with the thiol residues present on the glycoproteins of mucus. For this reason the nanoparticles derived from these chitosan derivatives were themselves endowed with mucoadhesivity. The thiolated nanoparticles formed by ionotropic gelation with TPP were stabilized via oxidation of thiols with H2O2 which formed interchain disulfide bonds. These would bestow gastroresistance on the particles, which would be particularly appropriate in case of oral administration of the nanoparticle formulation. However the presence of some non-oxidized thiols on the nanoparticle surface was needed to confer enhanced mucoadhesivity on such a surface. This goal was actually achieved by Bernkop-Schnurch et al. [21]. These authors also studied the crosslinker effect on nanoparticle size. Under similar preparation conditions, sizes in the 200-300 nm range were obtained with TPP as the crosslinker, whereas sizes beyond the micron resulted using Na2SO4.
2.2. Gelation from polyelectrolyte complex (PEC) formation
This method involved ionotropic gelation, just as described in the preceding section, only in the present case the crosslinker was a polyanionic polymer with charges opposite to those of the chitosan derivative, with which it formed a PEC. To this purpose
2.3. Polymer-drug complexes
Some negatively charged active principles, such as insulin or gene drugs, when mixed with cationic chitosan derivatives in adequate proportions spontaneously formed nanoparticulate dispersions of insoluble complexes [29-34]. TMC nanoparticles obtained by ionotropic gelation with TPP in the presence of insulin were compared with nanoparticles obtained by PEC formation between TMC and insulin. In the latter instance higher encapsulation efficiency and zeta potential (positive), and smaller particle size were observed, which is particularly appropriate for particle internalization into cells. In addition, a higher stability in simulated intestinal fluid (pH 6.8) of the nanocomplex compared to the nanoparticles prepared with TPP resulted [31,32].
2.4. Self-assembly
Amphiphilic derivatives of chitosan in aqueous solution were found, at a critical aggregation concentration (CAC), to spontaneously arrange into nanoparticles of sizes in between 100-400 nm. Such derivatives were prepared by connecting hydrophobic structures to the chitosan or glycol chitosan backbone via the amino group of the chitosan repeating unit. Examples of the above amphiphilic derivatives are the following: glycol chitosan-5β-cholanic acid conjugate [35-40]; palmitoyl chitosan [41]; palmitoyl glycol chitosan [42]; oleoyl chitosan [43]. Other amphiphiles were prepared from chitosans bearing fixed positive charges, in the case of quaternary ammonium palmitoyl glycol chitosan [42], or negative fixed charges, as in the case of linoleic acid-modified
The CAC for the hydrophobically modified chitosan derivatives is usually in the μM range, whereas the CMC of small-molecular weight surfactants is in the mM range. This is one of the most important characteristics of amphiphilic polymers, pointing to stability of the self-aggregates in dilute conditions, such as those the nanoparticles are supposed to encounter after administration to the organism. The CAC values of these polymers have been found to decrease with increasing hydrophobic content of derivatives [44]. In fact, the nanoparticles formed from these chitosan derivatives are characterized by a core-shell structure, i.e., a hydrophobic core in a hydrophilic shell. The drug incapsulation method was chosen on the basis of the hydrophilic or hydrophobic nature of the drug. With hydrophobic drugs the solution of polymer and drug in a water-miscible organic solvent was mixed with an aqueous medium and the organic solvent was cleared away by dialysis or evaporation [36,39,40]. Hydrophobic drugs having a fair water solubility and polar drugs have been loaded into nanoparticles via direct addition to the aqueous polymer dispersion [41,42,44,45]. The non-encapsulated drug has been separated by ultracentrifugation, filtration or dialysis.
3. Interactions with cells and tissues
3.1. Quaternized derivatives
Chitosan has been found to open the tight junctions connecting epithelial cells, through an interaction of its positively charged amino groups with negatively charged sites in the tight junctions, thereby promoting paracellular transepithelial absorption of drugs, peptides and proteins [10,46-57]. The major drawback of unmodified chitosan as an absorption promoter is its insolubility at physiological neutral pH. Therefore the primary amino groups of its repeating units have been quaternized to bestow fixed, pH-independent positive charges on the polymer, thus making it soluble and active as an absorption promoter at physiological pH. In fact, TMC was found to act as an enhancer of drug, peptide and protein permeability across intestinal, nasal, buccal, ocular epithelium [47, 58-66]. TMC was shown to promote not only paracellular but also transcellular drug absorption [66]. Other quaternized chitosan derivatives, namely,
Particles in the nanosize range have resulted from the interaction of quaternized chitosans with polyanions. Proteins or macromolecular drug models have been encapsulated in these nanoparticles. Ovalbumin (OVA) was encapsulated in nanoparticles, obtained by ionotropic gelation of TMC with TPP, and studied as a nasal delivery system for proteins [19]. No cytotoxicity of nanoparticles on Calu-3 cells, a model of human respiratory function, was evidenced, whereas a partially reversible cilio-inhibiting effect on the ciliary beat frequency of chiken trachea was observed. Confocal laser scanning microscopy (CLSM) of nasal epithelia and nasal associated lymphoid tissue (NALT), incubated with nanoparticles loaded with fluorescein-labelled albumin, showed the presence of fluorescent nanoparticles throughout the cytoplasm of these cells, indicating the transport of albumin-associated TMC/TPP nanoparticles across the nasal mucosa. These findings led the authors to point to these nanoparticles as a potential delivery system for transport of proteins through the nasal mucosa
Other authors studied similar TMC/TPP nanoparticles, loaded with fluorescein isothiocyanate dextran, molecular weight 4400 Da (FD4), as a model of macromolecular drugs [17]. In analogy with the free TMC, the TMC/TPP nanoparticles exhibited the property of opening the tight junctions between cells in the Caco-2 monolayer in vitro and the rat intestinal epithelium ex vivo, thus promoting the permeation of FD4 across the two epithelium models. The nanoparticles also shared, with the free TMC, the property of adhering to the intestinal mucosa. Using CLSM, Sandri et al. [17] showed internalization of their nanoparticles into Caco-2 cells and excised rat jejunum tissue.
Nanoparticles encapsulating fluorescein-labelled bovine serum albumin (BSA) were obtained by ionotropic gelation of alginate-modified TMC with TPP [16]. According to the authors the transport of alginate-modified TMC nanoparticles across the Caco-2 cell in vitro model of gastrointestinal (GI) epithelium was more efficient than that produced by non-modified TMC nanoparticles. However, alginate modification barely had any effect on the trans-epithelial electrical resistance or on paracellular protein transport. Then the hypothesis was made that alginate modification facilitated nanoparticle transport across the Caco-2 monolayer by the transcellular route (transcytosis) by virtue of a reduction of particle size to 100-200 nm (16). The supposedly permeated nanoparticles were assayed by measuring the fluorescence of fluorescein-labelled BSA, which was assumed to be completely associated with the particles. Similar nanoparticles as the above were loaded with urease, a vaccine protein against
OVA-loaded nanoparticles have been prepared from TMC using unmethylated CpG DNA as adjuvant and crosslinker, in place of TPP, for nasal vaccination in mice [15]. TMC/CpG/OVA showed similar physical properties as TMC/TPP/OVA in terms of particle size, zeta-potential and antigen release characteristics, but TMC/CpG/OVA induced a 10-fold higher IgG2a response than TMC/TPP/OVA, and a strong humoral and Th1 type cellular immune responses after nasal vaccination [15].
Nanoparticles derived from the polyelectrolytic complexation of TMC by the polyanionic mono-
Insulin was formulated into nanoparticles formed from quaternized chitosans such as TMC or DEMC via either ionotropic gelation with TPP, or polyelectrolyte complexation by the polyanionic insulin. The PEC method resulted in higher insulin loading efficiency and nanoparticle zeta-potential [31].
Similar nanoparticulate systems loaded with insulin were prepared from other quaternized chitosans, namely,
Poly(γ-glutamic acid) was used by Mi et al. [25] as the anionic polyelectrolyte complexing agent to prepare nanoparticles from TMC by the PEC method, for the oral delivery of insulin. According to the authors insulin was transported across the Caco-2 cell in vitro model of GI epithelium via the paracellular route. In fact, CLSM confirmed the opening of the tight junctions between cells caused by the nanoparticles. The authors propose a mechanism whereby the orally administered nanoparticles with mucoadhesive TMC on their surfaces may adhere and infiltrate into the intestinal mucus, mediate the opening of tight junctions between enterocytes, undergo disintegration, and release insulin, which would permeate through the paracellular pathway to the bloodstream. This hypothesis is contrasting with that, proposed by Chen et al. [16], of protein being carried by TMC/alginate/TPP nanoparticles across the Caco-2 monolayer by transcytosis.
TMC was modified with the specific ligand CSKSSDYQC peptide (CSK) to prepare ionotropically crosslinked TMC-CSK/TPP nanoparticles, loaded with fluorescein isothiocyanate (FITC)-labelled insulin, targeted to the mucus-producing goblet cells [45]. In transport studies across Caco-2/HT29-MTX co-cultured cell monolayer, simulating mucus-producing intestinal epithelium, the CSK modification showed enhanced drug transport ability, even if the target recognition was partially affected by mucus. In pharmacological and pharmacokinetic studies in diabetic rats, the orally administered CSK-modified nanoparticles produced a stronger hypoglycemic effect than the unmodified ones, prompting the authors to state that the former were sufficiently effective as goblet cell-targeting nanocarriers for oral delivery of insulin.
An oral delivery system for paclitaxel, a mitotic inhibitor used in cancer chemotherapy, was devised by encapsulating the drug in
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TMC-CSK | TPP | insulin | 200-350 | 3-10 | [14] |
HTCC |
o/w/o double emulsion | paclitaxel | 130 | 21 | [72] |
TMC | cisplatin-hyaluronate | cisplatin | 450 | 45 | [27] |
TMC | TPP | OVA | 300 | 20 | [15] |
TMC | MCC | tetanus toxoid, FITC-BSA | not reported | not reported | [24] |
HTCC | poly(aspartic acid) | BSA | 200-300 | 55 | [26] |
TMC | poly(γ-glutamic acid) | insulin | 100 | 30 | [25] |
TEC, DMEC | insulin | insulin | 200 | 25 | [30] |
TMC, DEMC | TPP, insulin | insulin | 250 | 25 | [31] |
TMC | TPP | FITC-BSA | 300 | 14 | [16] |
TMC | TPP | OVA | 254-300 | 20-61 | [19] |
TMC | TPP | FD4 | 200 | - | [17] |
3.2. Thiolated derivatives
The thiol groups immobilized on these polymers are supposed to give exchange reactions with disulfide bonds within the mucus, or oxidation reactions with cysteine-rich subdomains of mucus glycoproteins [73, 74], both resulting in the formation of disulfide bonds between thiolated chitosan derivatives and the mucus, which improve the polymer mucoadhesivity. Nanoparticles prepared from this type of chitosan derivatives were supposed to be themselves mucoadhesive, and hence, apt to make nanocarriers for oral drug delivery. In fact, enhanced mucoadhesive properties of nanoparticles prepared by gelation of chitosan-
Another thiolated chitosan derivative, chitosan-4-thiobutylamidine (chitosan-TBA) was used by Bernkop-Schnürch et al. [22] to develop a mucoadhesive nanoparticulate delivery system. The polymer was first crosslinked ionotropically by TPP, followed by stabilization of the resulting nanoparticles via formation of inter- and intrachain disulfide bonds by thiol oxidation with H2O2. Subsequently, TPP was removed by dialysis. The covalently crosslinked particles would not disintegrate in the acidic medium of the stomach. The adhesion to porcine intestinal mucosa was studied after incorporation of fluorescein diacetate into nanoparticles. The more thiol groups were oxidized, the lower was the nanoparticle mucoadhesivity, nevertheless, even when as much as 90% of all thiols were oxidized the mucoadhesivity of chitosan-TBA nanoparticles was twice as high as that of unmodified chitosan nanoparticles.
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Glycol chitosan coupled with thioglycolic acid (TGA) was ionotropically gelled with TPP to yield nanoparticles, which showed a twofold increase in mucoadhesion to lung tissue after intra-tracheal administration to rats as compared to non-thiolated nanoparticles. Biocompatibility of nanoparticle formulations with lung tissue was demonstrated. Calcitonin-loaded glycol chitosan and glycol chitosan-TGA nanoparticles resulted in a pronounced hypocalcemic effect for at least 12 and 24 h and a bioavailability of 27 and 40%, respectively [20].
Verheul et al. [28] used the thiol groups of thiolated TMC to spontaneously form interchain disulfide crosslinks with the thiols of thiolated hyaluronic acid (HA), after ionic gelation. OVA-loaded stabilized TMC-S-S-HA nanoparticles demonstrated higher immunogenicity than not stabilized particles, indicated by higher IgG titers, in nasal and intradermal vaccination.
Besides showing enhanced mucoadhesivity and cell penetration properties, nanoparticles made of thiolated chitosans have appeared highly effective as gene delivery systems. Thiolated derivatives, prepared from 33-kDa chitosan by coupling with TGA, formed nanocomplexes with plasmid DNA encoding green fluorescent protein (GFP), that were able to bind and protect plasmid DNA from Dnase I digestion. Thiolated chitosan/DNA nanocomplexes induced higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulfide-crosslinked thiolated chitosan/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Intranasal administration of crosslinked thiolated chitosan/DNA nanocomplexes to mice yielded gene expression that lasted at least 14 days [34].
Nanoparticles containing the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated, based on a thiolated chitosan conjugate, chitosan-TGA, crosslinked by thiol oxidation with H2O2 to form disulfide crosslinks. Transfection of nanoparticles in Caco-2 cells led to increased protein expression compared to unmodified chitosan nanoparticles. Red blood cells lysis tests provided evidence for no cytotoxicity of nanoparticles. On the basis of their experimental results the authors stated that their crosslinked thiolated chitosan nanoparticles showed the potential for being used as a non-viral vector system for gene therapy [33].
3.3. Amphiphilic derivatives
Amphiphilic derivatives resulted when hydrophobic structures were attached to the hydrophilic chitosan backbone. In aqueous milieu these derivatives would self-assemble into nanoparticles to attain thermodynamic stability. Nanoparticles derived from the self-assembly of amphiphilic derivatives were often intended for cancer therapy. Glycol chitosan (hydrophilic)-cholanic acid (hydrophobic) conjugates self-assembled to form nanoparticles, the in vivo tissue distribution, time-dependent excretion and tumor accumulation of which were monitored in tumor-bearing mice by Park et al. [37]. The particles exhibited prolonged blood circulation time, decreased time-dependent excretion from the body, and increased tumor accumulation with increasing polymer molecular weight. The enhanced tumor targeting by nanoparticles made of high molecular weight glycol chitosan-cholanic acid was ascribed to a better in vivo stability, related to an improvement in blood circulation time [37].
Similar nanoparticles as the above, formed from glycol chitosan-cholanic acid conjugate, loaded with the anticancer drug camptothecin, exhibited significant antitumor effects and high tumor targeting ability towards MDA-MB231 human breast cancer xenografts subcutaneously implanted in nude mice. The significant antitumor efficacy of nanoparticles was ascribed to both their prolonged blood circulation and high accumulation in tumors through the EPR effect [39].
The cellular uptake mechanism and the intracellular fate of nanoparticles formed from glycol chitosan hydrophobically modified with cholanic acid have been reported [40]. These particles showed an enhanced distribution in the whole cells, compared to the parent hydrophilic glycol chitosan polymer. In vitro experiments with endocytic inhibitors suggested that the cellular uptake of these nanoparticles involved several distinct pathways, e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis. Such a property, along with low toxicity and biocompatibility suggested these hydrophobically modified glycol chitosan nanoparticles as a versatile carrier for the intracellular delivery of therapeutic agents [40].
A further hydrophobically modified chitosan derivative from which self-assembled nanoparticles were obtained was oleoyl chitosan. The toxicity profile of the relevant nanoparticles, evaluated in vitro via hemolysis test and MTT assay, was within acceptable limits. When loaded with the antitumor drug doxorubicin, oleoyl chitosan nanoparticles exhibited inhibitory rates on different human cancer cells (A549, Bel-7402, HeLa, and SGC-7901) significantly higher than the drug solution [43].
Folic acid was conjugated with
The ability of nanoparticles prepared by self-assembly of chitosan amphiphiles to promote oral absorption of hydrophobic and hydrophilic drugs in rats was recently investigated by Siew et al. [42], using quaternary ammonium palmitoyl glycol chitosan as the basic material. The nanoparticles were found to enhance the oral absorption (Cmax) of griseofulvin and cyclosporine A (hydrophobic) and, to a lesser extent, of ranitidine (hydrophilic). Hydrophobic drug absorption was facilitated by the nanomedicine by: (a) increasing the drug dissolution rate, (b) adhering to and penetrating the mucus layer, thus allowing intimate contact between the drug and the GI epithelium absorptive cells, and (c) enhancing transcellular drug transport. As for the absorption of the hydrophilic ranitidine, despite an 80% increase of Cmax there was no appreciable opening of tight junctions by the nanoparticles. No uptake of this type of nanoparticles by epithelial cells is reported [42].
quaternary ammonium palmitoyl glycol chitosan |
griseofulvin cyclosporin A ranitidine |
100-500 | not reported | [42] |
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none | 300-400 | 10 | [40] |
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doxorubicin | 150-200 | 10-20 | [75] |
glycol chitosan-5β-cholanic acid | none | 230-310 | 10-11 | [37] |
glycol chitosan-5β-cholanic acid | camptothecin | 250-350 | not reported | [39] |
oleoyl-chitosan |
doxorubicin | 250-350 | not reported | [43] |
4. Concluding remarks
Three families of chitosan derivatives have been synthesized and used to prepare nanoparticles for pharmaceutical application, namely, polycations obtained by introducing quaternary ammonium groups on the polymer backbone; thiolated derivatives, and amphiphilic derivatives obtained by attaching hydrophobic structures to the chitosan or glycol chitosan backbone. The nanoparticles prepared from the quaternary ammonium-chitosan derivatives, especially via the PEC formation method, have shown improved stability and physical properties (smaller size, higher zeta potential) compared to nanoparticles from unmodified chitosan. The thiolated derivatives offered the opportunity to stabilize the nanoparticles by covalent crosslinks formed from interchain thiol oxidation to disulfide, which made the particles stable in the GI environment. The critical aggregation concentration of the amphiphilic hydrophobically modified chitosan derivatives is usually very low, which implies stability of the self aggregates in dilute conditions, such as those encountered by the nanoparticles in the organism. The nanoparticulate systems prepared from chitosan derivatives have generally shown acceptable cytotoxicity. In accord with the known behavior of particles of a size smaller than 500 nm, they have shown endocytic uptake by cells. Smaller particles with higher zeta potential have shown more aptitude to endocytosis. Ionotropically crosslinked TMC nanoparticles are a potential vehicle for transport of proteins across mucosal epithelia, as they have been found to open the tight junctions between epithelial cells. Indeed, nanoparticles based on quaternized chitosan are a promising vehicle for the oral administration of insulin, especially if the chitosan derivative is conjugated with the specific ligand CSKSSDYQC peptide. Also interesting is the nanosystem based on the quaternary ammonium-chitosan conjugate HTCC, which was orally absorbed by the rat small intestine and subsequently accumulated in carcinoma tissue by the EPR effect. These results are particularly intriguing as they open the prospect of a targeted oral treatment of cancer by nanomedicine. Nanoparticles prepared from thiolated chitosan derivatives have shown a particular mucoadhesivity implying a suitability for making nanocarriers for transmucosal protein delivery. Also this type of nanoparticles have appeared highly effective as gene delivery systems and have shown the potential for being used as a non-viral vector system for gene therapy. Nanoparticles derived from the self-assembly of amphiphilic chitosan derivatives were often intended for cancer therapy. Glycol chitosan hydrophobically modified with cholanic acid yielded nanoparticles with comparatively high in vivo stability, responsible for a prolonged blood circulation time, which led to high accumulation in tumors through the EPR effect. This type of nanoparticles can be taken up by cells through distinct pathways, which points to this system as a versatile carrier for the intracellular delivery of therapeutic agents. Folic acid, conjugated with
Abbreviations
BSA Bovine serum albumin
CAC Critical aggregation concentration
CLSM Confocal laser scanning microscopy
CSK CSKSSDYQC peptide
DEMC
DMEC
EPR Enhanced permeability and retention effect
FD4 Fluorescein isothiocyanate dextran, molecular weight 4400 Da
FITC Fluorescein isothiocyanate
GFP Green fluorescent protein
GI Gastrointestinal
HA Hyaluronic acid
HTCC
LLC Lewis lung carcinoma
MCC Mono-
NAC
NALT Nasal associated lymphoid tissue
OVA Ovalbumin
PEC Polyelectrolyte complex
pSEAP Recombinant secreted alkaline phosphatase
TBA Thiobutyl amidine
TEC
TGA Thioglycolic acid
TMC N-trimethyl chitosan
TPP Sodium tripolyphosphate
References
- 1.
Blood compatibility and biodegradability of partially N-acylated chitosan derivatives. BiomaterialsLee K. Y Ha W. S Park W. H 1995 16 1211 - 2.
Muzzarelli RAA Human enzymatic activities related to the therapeutic administration of chitin derivative. Cellular and Molecular Life Scince1997 53 131 - 3.
Biodegradation and distribution of water-soluble chitosan in mice. BiomaterialsOnishi H Machida Y 1999 20 175 - 4.
Chitosan as a biomaterial. Artificial Cells and Artificial OrgansChandy T Sharma C. P 1990 18 1 - 5.
Chitosan solutions on in vitro and in vivo mucociliary transport rates inhuman turbinates and volunteers. Journal of Pharmaceutical SciencesAspeden T. J Mason J. D Jones N. S Lowe J Skaugrud O Illum L 1997 86 509 - 6.
Chitosan as a novel nasal delivery system for peptide drugs. Pharmaceutical ResearchIllum L Farraj N. F Davis S. S 1994 11 1186 - 7.
Chitosan as a novel nasal delivery system for vaccines. Advanced Drug Delivery ReviewsIllum L Jabbal-gill I Hinchcliffe M Fisher A. N Davis S. S 2001 51 81 - 8.
Macromolecules as safe penetration enhancers for hydrophilic drugs-a fiction? Pharmaceutical Sciences Technology TodayJunginger H. E Verhoef J. C 1998 1 370 - 9.
Lueßen HL, de Boer AG, Verhoef JC, Lehr CM, Junginger HE. The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption: III. Effects of chitosan glutamate and carbomer on epithelial tight junctions in vitro. Journal of Controlled ReleaseBorchard G 1996 39 131 - 10.
Di Colo G Zambito Y, Burgalassi S, Nardini I, Saettone MF. Effect of chitosan and of N-carboximethylchitosan on intraocular penetration of topically applied ofloxacin. International Journal of Pharmaceutics2004 273 37 - 11.
Chitosan nanoparticles: a contribution to nanomedicine. Polymer IntenationalPeniche H Peniche C 2001 60 883 - 12.
Recent advances of chitosan nanoparticles as drug carriers. International Journal of NanomedicineWang J. J Zeng Z. W Xiao R. Z Xie T Zhou G. L Zhan X. R Wang S. L 2011 6 765 - 13.
Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers Journal of Polymer ScinceCalvo P Remunan-lopez C Vila-jato J. L Alonso M. J 1997 63 125 - 14.
Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport. BiomaterialsJin Y Song Y Zhu X Zhou D Chen C Zhang Z Huang Y 2012 33 1573 - 15.
Dual role of CpG as immune modulator and physical crosslinker in ovalbumin loaded N-trimethyl chitosan (TMC) nanoparticles for nasal vaccination. Journal of Controlled ReleaseSlütter B Jiskoot W 2010 148 117 - 16.
In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery. Intenational Journal of PharmaceuticsChen F Zhang Z. R Yuan F Qin X Wang M Huang Y 2008 349 226 - 17.
Di Colo G, Caramella C. Nanoparticles based on N-trimethylchitosan: Evaluation of absorption properties using in vitro (Caco-2 cells) and ex vivo (excised rat jejunum) models. European Journal of Pharmaceutics and BiopharmaceuticsSandri G Bonferoni M. C Rossi S Ferrari F Gibin S Zambito Y 2007 65 68 - 18.
Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro. International Journal of Biological MacromoleculesDehousse V Garbacki N Jaspart S Castagne D Piel G Colige A Evrard B 2010 46 342 - 19.
Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system. Journal of Controlled ReleaseAmidi M Romeijn S. G Borchard G Junginger H. E Hennink W. E Jiskoot W 2006 111 107 - 20.
Nanoparticles of glycol chitosan and its thiolated derivative significantly improved the pulmonary delivery of calcitonin. International Journal of PharmaceuticsMakhlof A Werle M Tozuka Y Takeuchi H 2010 397 92 - 21.
Development of a novel method for the preparation of submicron particles based on thiolated chitosan. European Journal of Pharmaceutics and BiopharmaceuticsBernkop-schnurch A Heinrich A Greimel A 2006 63 166 - 22.
Thiomers: Preparation and in vitro evaluation of a mucoadhesive nanoparticulate drug delivery system. Intenational Journal of PharmaceuticsBernkop-schnürch A Weithaler A Albrecht K Greimel A 2006 317 76 - 23.
Chitosan-NAC nanoparticles as a vehicle for nasal absorption enhancement of insulin. Journal of Biomedical Materials Research Part B: Applied BiomaterialsWang X Zheng C Wu Z Teng D Zhang X Wang Z Li C 2009 88 150 - 24.
TMC-MCC (N-trimethyl chitosan-mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines. European Journal of Pharmaceutical SciencesSayin B Somavarapu S Li X. W Sesardic D Senel S Alpar O. H 2009 38 362 - 25.
Oral delivery of peptide drugs using nanoparticles self-assembled by poly(γ-glutamic acid) and a chitosan derivative functionalized by trimethylation. Bioconjugate ChemistryMi F. L Wu Y. Y Lin Y. H Sonaje K Ho Y. C Chen C. T Juang J. H Sung H. W 2008 19 1248 - 26.
Quaternized chitosan (QCS/poly (aspartic acid) nanoparticles as a protein drug-delivery system. Carbohydrate ResearchWang T. W Xu Q Wu Y Zeng A. J Li M Gao X 2009 344 908 - 27.
Preparation, characterisation and preliminary antitumour activity evaluation of a novel nanoparticulate system basedon a cisplatin-hyaluronate complex and N-trimethyl chitosan. Invest New DrugsCafaggi S Russo E Stefani R Parodi B Caviglioli G Sillo G Bisio A Aiello C Viale M 2011 29 443 - 28.
Covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination. Journal of Controlled ReleaseVerheul R. J Slütter B Bal S. M Bouwstra J. A Jiskoot W Hennink W. E 2011 156 46 - 29.
Preparation and characterization of insulin nanoparticles using chitosan and its quaternized derivatives. NanomedicineBayat A Larijani B Ahmadian S Junginger H. E Rafiee-tehrani M 2008 4 115 - 30.
Nanoparticles of quaternized chitosan derivatives as a carrier for colon delivery of insulin: Ex vivo and in vivo studies. Intenational Journal of PharmaceuticsBayat A Dorkoosh F. A Dehpour A. R Moezi L Larijani B Junginger H. E Rafiee-tehrani M 2008 356 259 - 31.
Sadeghi AMM Dorkoosh FA, Avadi MR, Saadat P, Rafiee-Tehrani M, Junginger HE. Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods. International Journal of Parmaceutics2008 355 299 - 32.
Peroral delivery of insulin using chitosan derivatives: A comparative study of polyelectrolyte nanocomplexes and nanoparticles. International Journal of PharmaceuticsJintapattanakit A Junyaprasert V. B Mao S Sitterberg J Bakowsky U Kissel T 2009 342 240 - 33.
Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cell culture. NanotechnologyMartien R Loretz B Sandbichler A. M Bernkop-schnürch A 2008 - 34.
Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery. Pharmaceutical ResearchLee D Zhang W Shirley S. A Kong X Hellermann G. R Lockey R. F Mohapatra S. S 2007 24 157 - 35.
Self-assembled nanoparticles nanoparticles containing hydrophobically modified glycol chitosan for gene delivery. Journal of Controlled ReleaseYoo H. S Lee J. E Chung H Kwon I. C Jeong S. Y 2005 103 235 - 36.
Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. Journal of Controlled ReleaseKim J. H Kim Y. S Kim S Park J. H Kim K Choi K Chung H Jeong S. Y Park R. W Kim I. S Kown I. C 2006 111 228 - 37.
Effect of polymer molecular weight on the tumor targeting characteristics of self-assembled glycol chitosan nanoparticles. Journal of Controlled ReleasePark K Kim J. H Nam Y. S Lee S Nam H. Y Kim K Park J. H Kim I. S Choi K Kim S. Y Kwon I. C 2007 122 305 - 38.
Self-assembled glycol chitosan nanoparticles for the sustained and prolonged delivery of antiangiogenic small peptide drugs in cancer terapy. BiomaterialsKim J. H Kim Y. S Park K Kang E Lee S Nam H. Y Kim K Park J. H Chi D. Y Park R. W Kim I. S Choi K Kwon I. C 2008 29 1920 - 39.
Hydrophobically modified glycol chitosan nanoparticles-encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy. Journal of Controled ReleaseMin K. H Park K Kim Y. S Bae S. M Lee S Jo H. G Park R. W Kim I. S Jeong S. Y Kim K Kwon I. C 2008 127 208 - 40.
Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. Journal of Controlled ReleaseNam H. Y Kwon S. M Chung H Lee S. Y Kwon S. H Jeon H Kim Y Park J. H Kim J Her S Oh Y. K Kwon I. C Kim K Jeong S. Y 2009 135 259 - 41.
Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer. BiomaterialChen K. J Chiu Y. L Chen Y. M Ho Y. C Sung H. W 2011 32 2586 - 42.
Enhanced oral absorption of hydrophobic and hydrophilic drugs using quaternary ammonium palmitoyl glycol chitosan nanoparticles. Molecular PharmaceuticsSiew A Le H Thiovolet M Gellert P Schatzlein A Uchegbu I 2012 9 14 - 43.
Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin. Nanomedicine: Nanotechnology, Biology, and MedicineZhang J Chen X. G Li Y. Y Liu C. S 2007 3 258 - 44.
Self-aggregated nanoparticles from linoleic acid modified carboxymethyl chitosan: Synthesis, characterization and application in vitro. Colloids and Surfaces B: BiointerfaceTan Y. L Liu C. G 2009 69 178 - 45.
Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport. BiomaterialsJin Y Song Y Zhu X Zhou D Chen C Zhang Z Huang Y 2012 33 1573 - 46.
Chitosans as absorption enhancers for poorly absorbable drugs. 1. Influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells. Pharmaceutical ResearchSchipper N. G Varum K. M Artusson P 1996 13 1686 - 47.
Lueßen HL, de Boer AG, Verhoef JC, Junginger HE. Chitosans for enhanced delivery of therapeutic peptides across intestinal epithelia: in vitro evaluation in Caco-2 cell monolayers. Internationa Journal of PharmaceuticsKotzé A. F De Leeuw B. J 1997 159 243 - 48.
Lueßen HL Rentel CO, Kotzé AF, Lehr CM, de Boer AG, Verhoef JC, Junginger HE. Mucoadhesive polymers in peroral peptide drug delivery. IV. Polycarbophil and chitosan are potent enhancers of peptide transport across intestinal mucosae in vitro. Journal of Controlled Release1997 45 15 - 49.
Lueßen HL de Leew BJ, Langmeÿer MWE, de Boer AG, Verhoef JC, Junginger HE. Mucoadhesive polymers in peroral peptide drug delivery. VI. Carbomer and chitosan improve the intestinal absorption of the peptide drug buserelin in vivo. Pharmaceutical Research1996 13 1668 - 50.
Chitosan as novel nasal delivery system for peptide drugs. Pharmaceutical ResearchIllum L Farraj N. F Davis J. J 1994 11 1186 - 51.
Chitosans as nasal absorption enhancers of peptides: comparison between free amine chitosans and soluble salts. International Journal of PharmaceuticsTengamnuay P Sahamethapat A Sailasuta A Mitra A. K 2000 197 53 - 52.
Enhancing effect of chitosan on nasal absorption of salmon calcitonin in rats: comparison with hydroxypropyl- and dimethyl-β-cyclodextrins. Intenational Journal of PharmSinswat P Tengamnuay P 2003 257 15 - 53.
Effect of chitosan on the intranasal absorption of salmon calcitonin in sheep. Journal of Pharmacy and PharmacologyHinchcliffe M Jabbal-gill I Smith A 2005 57 681 - 54.
Permeation-enhancing effects of chitosan formulations on recombinant hirudin-2 by nasal delivery in vitro and in vivo. Acta Pharmacologica SinicaZhang Y. J Ma C. H Lu W. L Zhang X Wang X. L Sun J. N Zhang Q 2005 26 1402 - 55.
Di Colo G Burgalassi S, Chetoni P, Fiaschi MP, Zambito Y, Saettone MF. Gel-forming erodible inserts for ocular controlled delivery of ofloxacin. International Journal of Pharmaceutics2001 215 101 - 56.
Schipper NGM Varum KM, Stemberg P, Ocklind G, Lennernäs H. Chitosans as absorption enhancers of poorly absorbable drugs. 3: Influence of mucus on absorption enhancement. European Journal of Pharmaceutical Sciences1999 8 335 - 57.
Enhancement of bronchial octreotide absorption by chitosan and N-trimethyl chitosan shows linear in vitro/in vivo correlation. Journal of Controlled ReleaseFlorea B. I Thanou M Junginger H. E Borchard G 2006 110 353 - 58.
Di Colo G, Caramella C. Buccal penetration enhancement properties of N-trimethyl chitosan: Influence of quaternization degree on absorption of a high molecular weight molecule. International Journal of PharmaceuticsSandri G Rossi S Bonferoni M. C Ferrari F Zambito Y 2005 297 146 - 59.
Junginger HE. N-trimethyl chitosan chloride as a potential absorption enhancer across mucosal surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2). Pharmaceutical ResearchKotzé A. F Luessen H. L De Leew B. J De Boer B. G Verhoef J. C 1997 14 1197 - 60.
Enhancement of paracellular drug transport with highly quaternized N-trimethyl chitosan chloride in neutral environments: in vitro evaluation in intestinal epithelial cells (Caco-2). Journal of Pharmaceutical SciencesKotzé A. F Thanou M Luessen H. L De Boer B. G Verhoef J. C Junginger H. E 1999 88 253 - 61.
Intestinal absorption of octreotide: N-trimethyl chitosan chloride (TMC) ameliorates the permeability and absorption properties of the somatostatin analogue in vitro and in vivo. Journal of Pharmaceutical SciencesThanou M Verhoef J. C Marbach P Junginger H. E 2000 89 951 - 62.
Junginger HE. N-trimethyl chitosan chloride (TMC) improves the intestinal permeation of the peptide drug buserelin in vitro (Caco-2 cells) and in vivo (rats), Pharmaceutical ResearchThanou M Florea B. I Langemeyer M. W Verhoef J. C 2000 17 27 - 63.
Effect of the degree of quaternization of N-trimethyl chitosan chloride on absorption enhancement: in vivo evaluation in rat nasal epithelia. Internationa Journal of PharmaceuticsHamman J. H Stander M Kotzé A. F 2002 232 235 - 64.
Enhancement of bronchial octreotide absorption by chitosan and N-trimethyl chitosan shows linear in vitro/in vivo correlation. Journal of Controlled ReleaseFlorea B. I Thanou M Junginger H. E Borchard G 2006 110 353 - 65.
Di Colo G, Burgalassi S,. Zambito Y, Monti D, Chetoni P. Effects of different N-trimethyl chitosans on in vitro/in vivo ofloxacin transcorneal permeation. Journal of Pharmaceutical Sciences2004 93 2851 - 66.
Di Colo G. Effects of N-trimethylchitosan on transcellular and paracellular transcorneal drug transport. European Journal of Pharmaceutics and BiopharmaceuticsZambito Y Zaino C 2006 64 16 - 67.
Kotzé AF. N-trimethyl chitosan chloride: optimum degree of quaternization for drug absorption enhancement across epithelial cells. Drug Development and Industrial PharmacyHamman J. H Schultz C. M 2003 29 161 - 68.
Optimized synthesis and characterization of N-triethylchitosan. Journal of Bioactive Compatible PolymersAvadi M. R Zohuriaan-mehr M. J Younessi P Amini M Rafiee-tehrani M Shafiee A 2003 18 469 - 69.
Sadeghi AMM, Avadi MR, Amini M, Rafiee-Tehrani M, Shafiee A, Majlesi R, Junginger HE. Synthesis of N,N-dimethyl N-ethylchitosan as a carrier for oral delivery of peptide drugs. Journal of Bioactive Compatible PolymersBayat A 2006 21 433 - 70.
Sadeghi AMM, Tahzibi A, Bayati KH, Pouladzadeh M, Zohuriaan-mehr MJ, Rafiee-Tehrani M. Diethylmethyl chitosan as antimicrobial agent: synthesis, characterization and antibacterial effects. European Journal of PolymersAvadi M. R 2004 40 1355 - 71.
The use of mucoadhesive polymers in ocular drug delivery. Advanced Drug Delivery ReviewLudwig A 2005 57 1595 - 72.
Porous quaternized chitosan nanoparticles containing paclitaxel nanocrystals improved therapeutic efficacy in non-small-cell lung cancer after oral administration. BiomacromoleculesLv P. P Wei W Yue H Yang T. Y Wang L. Y Ma G. H 2011 12 4230 - 73.
Thiolated polymers- thiomers: development and in vitro evaluation of chitosan-thioglycolic acid conjugates. BiomaterialsKast C. E Bernkop-schnurch A 2001 22 2345 - 74.
Thiolated polymers: evidence for the formation of disulfide bonds with mucus glycoproteins. European Journal of Pharmaceutics and BiopharmaceuticsLeitner V. M Walker G. F Bernkop- Schnürch A 2003 56 207 - 75.
In vitro evaluation of folic acid modified carboxymethyl chitosan nanoparticles loaded with doxorubicin for targeted delivery. Journal of Materials Science: Materials in MedicineSahu S Mallick S. K Santra S Maiti T. K Ghosh S. K Pramanik P 2010 21 1587