Preparation of chitosan nanoparticles by nanoprecipitation and their ability as a drug nanocarrier

Abstract

Polysaccharide-based nanoparticles represent a very promising drug delivery platform, particularly for the transmucosal delivery of bioactive macromolecules. Thus, the aim of this paper is to revisit the nanoprecipitation processes for preparing chitosan nanoparticles and to evaluate the influence of the process parameters on their characteristics. Chitosan was dissolved in water as N-(methylsulfonic acid) chitosan or directly in aqueous acetic acid. Methanol was used as the nonsolvent diffusing phase. Nanoparticles became smaller as the polymer concentration decreased or the nonsolvent to solvent volume ratio increased. Particles prepared in acidic media are slightly larger than those precipitated from N-(methylsulfonic acid) chitosan. Replacement of methanol by water in the suspension medium resulted in a notorious increase in their size. On the other hand, very little additions of Tween-80 to the nonsolvent phase render smaller nanoparticles, with a similar mean-size values. Nanoparticles precipitated in methanol have roughly the same dimensions, regardless of the ionic strength of the chitosan solution. These chitosan nanoparticles have good association and loading efficiency values of a model substance showing their ability as a nanocarrier for drug delivery systems.

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1. Introduction

During the past few years, there has been considerable interest in the use of polymeric nanoparticles as carriers of bioactive molecules. These systems have the ability to cross biological barriers and to protect the active molecules such as peptides, proteins, oligonucleotides or genes from degradation in the biological milieu, while delivering the bioactive molecules to the proper specific site.¹ Nanoparticles have been described as colloidal solids with a diameter ranging from 1 to 1000 nm consisting of macromolecules on which drugs may be entrapped, adsorbed or chemically-bound.²

There is an increasing interest in investigating these submicron particles due to their potential capacity for carrying drugs, targeting systems and overcoming the typical problems of conventional drug delivery systems due to the stability, dissolution, gastrointestinal mucosa irritation or the disagreeable organoleptic properties of the active substances used. Consequently, the preparation method is a key step for ensuring that particles behave according to the intended use.³ The properties of polymeric nanoparticles have to be optimized depending on the particular application. In order to achieve the properties of interest, the mode of preparation plays a vital role. Thus, it is highly advantageous to have preparation techniques at hand to obtain polymeric nanoparticles with the desired properties for a particular application. Although some information regarding preparation techniques of polymeric nanoparticles is available, it is scattered in the literature and restricted to a few areas.²

Two main physicochemical routes have been reported for the preparation of nanoparticles. The first technique comprises the polymerization of monomers, while the second relies on the dispersion of previously-synthesized polymers. Typical examples of this group comprise ionic gelation, salting-out, emulsification-diffusion and nanoprecipitation procedures.^4,5

Nanoprecipitation, also known as solvent-displacement technique, provides numerous advantages over other methods. It is rapid and easy to carry out. Nanoparticle formation is instantaneous and the procedure takes place in one-step approach.⁶ However, nanoprecipitation involves complex hydrodynamic and phase separation processes. The mechanism of nanoparticle formation can be explained in terms of interphase turbulence between two liquid phases involving flow processes, diffusion and surface interactions at a non-equilibrium stage.⁷ The formation of submicron particles depends on the combination of operating conditions, the composition of the organic and aqueous phases (since it determines their physicochemical properties) and the physicochemical interactions between phases. The extent of their participation is still unclear, though it appears that one prevails over another depending on their interrelationship.^3,8

During the nanoparticle formation process the bioactive molecule is dissolved, entrapped, encapsulated or bounded to a matrix. Such a matrix can be obtained from various natural or synthetic polymers including, for example, anionic copolymers based on methacrylic acid and ethyl acrylate,⁹ poly-D-L-lactide-co-glicolide polymers,^6,7 dextran hydrophobic derivatives,¹⁰ poly-D-L-lactic acid,¹¹ polyvinyl alcohol,¹² chitosan-poly-D-L-lactic acid (PLA),¹³ among others.

Polysaccharide-based nanoparticles nowadays represent a very promising drug delivery platform, particularly for the transmucosal delivery of bioactive macromolecules. Their usefulness relies on a number of interesting properties, namely, muco- and bioadhesiveness, a high capacity to associate and release therapeutic macromolecules in their bioactive form, as well as their ability to enhance the transport of bioactive compounds across well-organized epithelial barriers, such as the ocular, nasal and intestinal routes.¹⁴

Chitosan is obtained from the chemical or enzymatic deacetylation of chitin, the major polysaccharide component of crustacean exoeskeleton. Chitin is comprised by chains with different proportions of glucosamine and N-acetylglucosamine.¹⁵ Chitosan has been shown to be a biocompatible, biodegradable and non-toxic natural polymer which possesses various desirable properties for the manufacture of drug-release systems.¹⁶ Chitosan has the ability to interrupt the intercellular connections thus increasing their permeability.¹⁷ Furthermore, Huang et al. assessed the mechanism of cellular penetration by chitosan molecules and chitosan nanoparticles and found that they easily penetrate A549 cells without cytotoxic properties. They concluded that the degree of deacetylation had more influence than the molecular weight on cellular penetration.¹⁸

Goycoolea et al. obtained a new type of hybrid chitosan–tripolyphosphate–alginate nanoparticle generated by the concomitant ionic cross-linking of chitosan to tripolyphosphate, this system was able to associate insulin, with efficiencies up to 50.7% and release it in vitro.¹⁴ Wilson et al. prepared tacrine-loaded chitosan nanoparticles by spontaneous emulsification. Nanoparticles prepared by this method showed good drug-loading capacity and the in vitro release studies indicated that after the initial burst, all the drug-loaded batches provided a continuous and slow release of the drug.¹⁹

In a recent report, Kafshgari et al. prepared chitosan nanoparticles using a reverse micellar system composed of cetyl trimethyl ammonium bromide as a surfactant, isooctane as a solvent, and 1-hexananol as a co-solvent. They found that the size of nanoparticles decreased with increasing the volumetric ratio of co-solvent/solvent. The polymer concentration was the main parameter affecting the size of nanoparticles.²⁰ This research group has also studied the role of calcium alginate to reinforce chitosan–tripolyphosphate nanoparticles and concluded that using higher concentrations of alginate resulted in smaller nanoparticles but resulted in lower albumin entrapment efficiency.²¹

The aim of this paper is to revisit the nanoprecipitation processes for preparing chitosan nanoparticles, to evaluate the influence of the preparation conditions on their characteristics and to assess their capacity as drug nanocarrier.

2. Experimental

2.1. Materials

A commercially available medical grade chitosan sample was supplied by NovaMatrix (Protasan UP B 80/20, Batch BP-0806-03). Its viscosity-average molecular weight was 1.1 × 10⁵, estimated at 25 °C in 0.3 M acetic acid/0.2 M sodium acetate²² and the degree of N-acetylation (DA = 0.178) was determined by ¹H NMR spectroscopy. According to the supplier, this sample has the following characteristics: ash content 0.06%, protein content 0.13% and endotoxins < 43 EU g⁻¹.

Chitosan hydrochloride was prepared in situ by dissolving chitosan in water after carefully adding a stoichiometric amount of hydrochloric acid.

All other reagents and solvents (Sigma-Aldrich) were used without further purification. Experiments were carried out with distilled water (conductivity lower than 3 μS cm⁻¹).

2.2. Nanoparticle preparation

The polymer was dissolved in a suitable solvent at concentrations from 0.5 to 2 mg mL⁻¹ to form the diffusing phase. This phase (with volumes typically ranging from 1 to 10 mL) was then added to the dispersing phase (25 or 40 mL) by means of a needle positioned two centimeters above the surface at 0.86 mL min⁻¹ using a peristaltic pump, under moderate magnetic stirring. This procedure allowed nonsolvent to solvent volume ratios between 5 : 1 to 40 : 1. The dispersing phase was methanol, an organic solvent miscible with water in which the polymer is insoluble – the nonsolvent – optionally containing a nonionic surfactant (Tween 80 at a concentration 0.05%). The freshly formed nanoparticles were characterized by various means described hereafter. The above procedure was used, unless otherwise stated.

2.3. Dynamic Light Scattering (DLS)

Particle size and polydispersity were determined by Dynamic Light Scattering (DLS). A digital correlation system ALV-5000 (ALV-GmbH, Langeln, Germany) equipped with an argon laser (30 mW; λ₀ = 632 nm) was used at 25 ± 0.1 °C.

The refractive index was measured in a refractometer Model RE4OD (Mettler Toledo). The hydrodynamic radius R_H, was obtained at an incident angle of 90° using the Stokes–Einstein equation:

D₀ = k_BT/6πηR_H,

where D₀ is the diffusion coefficient, k_B is the Boltzmann constant, T is the absolute temperature and η is the viscosity of the solvent.

2.4. Atomic Force Microscopy (AFM)

The experiments were conducted at room temperature in a non-contact mode using a JEOL microscope equipped with a silicon cantilever NSC15 from MikroMasch (MikroMasch, Portland, USA). A drop of chitosan nanoparticle suspension was placed on mica and dried under atmospheric conditions before proceeding for analysis. The experiment was carried out using an atomic force JEOL Microscope (Scanning Probe Microscope, Model JSPM 4210, Japan).

Height and cross-sectional size measurements were carried out from AFM images with WSxM software, version 4.0, from Nanotec Electronica S.L. (Madrid, Spain).²³

2.5. Scanning Electron Microscopy (SEM)

Five milliliters of each sample were placed in a 10 mL vial and sonicated for 20 minutes at 60 °C. Three drops of the sample were placed on the sample holder using highly conductive graphite paint as adhesive to fix the sample and allowed to dry for 24 hours. The morphology of the samples was then studied by scanning electron microscopy in a JEOL 6360LV microscope (JEOL Inc., Peabody, MA, USA) in high vacuum mode, operating at 20 kV and a spot size 11.

2.6. Nanoparticle entrapment capacity

Citral (3,7-dimethyl-2,6-octadienal) was chosen as a model drug to evaluate the entrapment capacity of these nanoparticles. With this purpose, nanoparticles were prepared as described in Section 2.2, but adding citral to the chitosan solution (chitosan 0.5 mg mL⁻¹ and citral 0.04–0.24 μg mL⁻¹). Nonsolvent to solvent volume ratio 10 : 1. The dispersing phase was methanol. Resulting citral-loaded nanoparticles were separated from the suspension by centrifugation at 15 000 RCF and 15 °C for 40 min.

The quantity of citral entrapped in the nanoparticles was calculated by the difference between the total citral added to the formation medium and the quantity of unbound citral remaining in the clear supernatant, measured by UV spectrophotometry at 226 nm. A standard curve of citral was prepared in a mixture of 2% aqueous acetic acid and methanol (volume ratio 1 : 10). Blank was made with the same solvent mixture.

The association and loading efficiencies of citral were calculated as follows:

3. Results and discussion

Chitosan is normally insoluble in water at neutral pH. Thus, it requires the addition of acid for solubility. Under these conditions, chitosan behaves as a cationic polyelectrolyte as the free amine groups are protonated giving rise to the corresponding salt. In recent years, Roberts has proposed a novel “solvent system”, which permits chitosan to be readily dissolved in water.²⁴ By this approach, chitosan reacts with sodium formaldehyde bisulfite in aqueous media and is converted into a strong anionic polyelectrolyte N-(methylsulfonic acid) chitosan, which chemical structure is Chit-NHCH₂SO₃Na. In our case, chitosan was dissolved in both, 2% aqueous acetic acid and in 3% sodium formaldehyde bisulfite.

Fig. 1 shows the average particle size obtained, when chitosan nanoparticles were prepared as N-(methylsulfonic acid) chitosan using polymer solutions at 0.5 and 2 mg mL⁻¹ and different nonsolvent to solvent ratios. It could be appreciated that the higher the polymer concentration, the greater the average particle size. This effect is particularly evident when nanoparticles were prepared from a chitosan solution with concentration of 2 mg mL⁻¹. In this case particles were markedly large ranging between 980 and 1900 nm. It is also noted that, regardless of the polymer concentration, there is a tendency in the mean size to slightly diminish with increases in the amount of nonsolvent during the precipitation. This behavior is apparent for both, chitosan dissolved in acetic acid and N-(methylsulfonic acid) chitosan in aqueous solution at neutral pH. In the same figure it could be perceived that under same experimental conditions (polymer concentration and nonsolvent to solvent ratio), particles prepared in acidic media are larger than those precipitated from N-(methylsulfonic acid) chitosan.

Fig. 1 Effect of type of chitosan solution, polymer concentration and nonsolvent to solvent volume ratio on the mean size of nanoparticles. 2 mg mL⁻¹ (squares), 0.5 mg mL⁻¹ (circles). N-(Methylsulfonic acid) chitosan (filled symbols) and 2% aqueous acetic acid (open symbols).

Fig. 2a and b show the DLS profiles, AFM images, and line-scan profiles of nanoparticles obtained from acetic acid and N-(methylsulfonic acid) chitosan, respectively. The differences in size and shape are evident. The sample previously dissolved in acetic acid gives almost spherical-shape particles with satisfactory size dispersion. These particles are seen as round objects with diameter of 290 nm and height of 13 nm, probably due to the drying effect. On the other hand, N-(methylsulfonic acid) chitosan particles are steeper in shape with diameter of 230 nm and height of 23 nm. They present a narrower size distribution.

Fig. 2 Effect of type of chitosan solution on the characteristics of nanoparticles (DLS distributions, AFM images and line scan profiles based on the scan along the lines A–A and B–B). Polymer concentration: 0.5 mg mL⁻¹; nonsolvent to solvent volume ratio: 40 : 1. (a) 2% aqueous acetic acid and (b) N-(methylsulfonic acid) chitosan.

Given the similarity in behavior of both nanoparticulate systems, thereafter the work was carried out only with chitosan dissolved in dilute acetic acid at 0.5 mg mL⁻¹. This is a cleaner and lower-cost process.

One of the drawbacks of preparing chitosan nanoparticles for biomedical purposes by this method is the presence of methanol in the suspension. In order to avoid it, an attempt was made to replace methanol by water. It was accomplished by a simple rotatory evaporation under vacuum at 55 °C and further re-suspended with water up to the original volume. This procedure was repeated four times for each sample. Fig. 3a shows the effect of this process on the characteristics of the final particles. There is a notorious increment in the particle size, irrespective of the nonsolvent to solvent volume ratio employed during the precipitation.

Fig. 3 Effect of replacing methanol by water in the suspension medium on the mean size of nanoparticles at different nonsolvent to solvent volume ratios. Original methanol suspension (filled circles), water suspension (open circles). Polymer concentration: 0.5 mg mL⁻¹ in 2% aqueous acetic acid. (a) Without addition of surfactant and (b) with Tween 80 added to methanol before the nanoprecipitation. Surfactant concentration: 0.05%.

The addition of surfactants to the nonsolvent phase has been reported to provide stability to nanoparticulate systems. Thus, Tween 80 was added to the methanol phase at a 0.05% concentration. Results from these experiments are summarized in Fig. 3b, where a decrease in particle size could be seen, and at the same time, a clear tendency to render the same particle size regardless of the amount of nonsolvent used for the precipitation. A similar behavior has been observed for other polymer nanoparticles prepared by nanoprecipitation,³ e.g., poly(D,L-lactide)/Pluronic F-68,²⁵ hydrophobic molecules²⁶ and starch/Tween 80.²⁷ Nevertheless there are some other systems in which the addition of surfactants has no effect on the particle size as is the case of poly(lactic-co-glycolic acid).⁶ It has also been recognized that surfactants improve the stability of nanoparticulate systems.²⁸ The apparent contradictory role of surfactant indicates that their effect depends of the type of specific surfactant-polymer binding. In our case, it should occur mainly through hydrogen-bonding interactions between oxyethylene moieties of the surfactant and hydroxyl groups of chitosan.

Re-suspended chitosan/Tween 80 system renders nanoparticles with diameters around 600–700 nm, spherical in shape and smooth surface as evidenced from the scanning electron microscopy (Fig. 4).

Fig. 4 Micrographs of re-suspended chitosan/Tween 80 system obtained by SEM. Polymer concentration: 0.5 mg mL⁻¹ in 2% aqueous acetic acid; nonsolvent to solvent volume ratio 40 : 1.

Frequently it is necessary to keep the nanoparticulate system during long periods and the most suitable way to achieve it is to freeze-dry the sample. With this aim, the influence of this process was also evaluated. Samples, in which methanol was replaced with water by the abovementioned procedure were lyophilized. Prior to being characterized, the dried sample was resuspended in the same volume of water and sonicated (five one-minute cycles, 42 kHz). There is a slight tendency to increase the mean size of the particles (Fig. 5). AFM image and line profile evidences that these particles are more irregular with sizes of 600 nm and height of 38 nm.

Fig. 5 Effect of the resuspension of freeze-dried nanoparticles in water on the mean size of nanoparticles. Polymer concentration: 0.5 mg mL⁻¹ in 2% aqueous acetic acid; nonsolvent to solvent volume ratio 40 : 1. Inside the graph is the AFM image and line scan profile of the resuspended nanoparticle based on the scan along the line A–A of the AFM image.

Fig. 6 summarizes the results obtained to assess the influence of the ionic strength of chitosan solutions, as well as the type of the nonsolvent used during the precipitation. The differences between nanoparticles obtained when using different nonsolvent phases are evident (left and right panels in Fig. 6). In general, nanoparticles prepared by precipitation in acetone result in sizes up to 6 μm, which are far from the nanoscale region. Galindo-Rodriguez et al. provide a serious discussion about the role of nonsolvent–solvent interaction parameter on the phase-separation processes that take place during the complex nanoprecipitation procedure.⁹ According to their analysis, the lower the χ_{organic solvent–water} value, the smaller the particle size during the nanoprecipitation. Even when our system was the opposite of theirs (organic soluble polymer precipitated in water), the χ_{water–organic solvent} keeps the same value and physical meaning. The acetone–water pair presents the higher value of the interaction parameter and renders the larger chitosan particles.

Fig. 6 Effect of the nonsolvent employed during nanoprecipitation on the mean size of nanoparticles, as indicated in the figure. Left-hand panel: chitosan dissolved in 2% aqueous acetic acid; right-hand panel: chitosan hydrochloride dissolved in water. Polymer concentration: 0.5 mg mL⁻¹; nonsolvent to solvent ratio 10 : 1 (if labeled, surfactant was dissolved in the nonsolvent phase at 0.05%).

It is known that the polymer–solvent interaction parameter characterizes the energy of interaction of polymer segments with solvent molecules within the Flory–Huggins theory. It is the only way to take into account the specific chemical nature and interactions between polymer and solvent. This parameter allows the thermodynamic treatment of the polymer–solvent mixing process, and also the phase separation phenomena.^29,30

A chitosan solution in 2% aqueous acetic acid has a high ionic strength as result of a substantial excess of acetic acid: the concentration of chitosan amine groups is 2.4 mM, while that of acetic acid is 333 mM. This condition screens the electrostatic repulsions between the ionic groups of chitosan and should induce a strong reduction of the hydrodynamic volume of the polyelectrolyte. Nevertheless, nanoparticles precipitated in methanol have roughly the same dimensions, regardless of the ionic strength of the chitosan solution (left and right panels in Fig. 6).

The value of χ for chitosan in 2% acetic acid has been experimentally estimated as −0.01, and it is interesting to note that the addition of salt does not change the value of χ and therefore, it does not affect the excluded volume interaction.³¹ According to our results, there are no differences between nanoparticles obtained from low or high ionic strength chitosan solution, using methanol as the nonsolvent phase. Even though there is no information about the precise role of the polymer–solvent interaction parameter on the nanoprecipitation, the coincidence between the behavior of the Flory's interaction parameter and the dimensions of nanoparticles at different salt concentrations is clearly seen.

Finally, as has already been discussed, the effect of the surfactant – reducing the particle size when using either methanol or acetone as the nonsolvent (Fig. 6) – is also clear.

In order to assess the capacity of these nanoparticles to entrap substances, citral-loaded nanoparticles were prepared. Citral is an important essential oil component that has showed appreciable antimicrobial effect against Gram-positive and Gram-negative bacteria as well as fungi. Even more, turmeric oil, an essential oil containing citral, has recently been demonstrated to retain its antriproliferative activity when included in chitosan-alginate nanocapsules.^32–34

Citral entraping experiments were carried out using methanol as the dispersing phase, using nonsolvent to solvent volume ratio 10 : 1 and different amounts of citral. Results are shown in Fig. 7. It is obvious that citral could be efficiently incorporated to all nanoparticle preparations, reaching association efficiencies around 88% for the formulation with the higher citral content. These association values are slightly greater than those obtained previously for nobiletin-loaded chitosan nanoparticles by our group³⁵ and are similar to the values obtained in chitosan/TPP nanoparticles when encapsulating some model drugs.^14,36,37

Fig. 7 Effect of the amount of the total citral added to the formation medium on the association efficiency (filled circles) and loading efficiency (open circles). Polymer concentration: 0.5 mg mL⁻¹ in 2% aqueous acetic acid. Nonsolvent to solvent volume ratio 10 : 1.

Regarding the loading efficiency values, the same tendency is observed when the amount of citral is increased. It was possible to reach citral content in the nanoparticles of up to 38%, meaning that these nanoparticles are composed by 62% polymer and 38% of drug. In general, it should be noted that the loading efficiency gives also similar values, as compared with chitosan nanoparticles prepared by ionotropic gelation with TPP,^14,36 but higher than those reached by nobiletin-loaded chitosan nanoparticles also prepared by nanoprecipitation in methanol.³⁵

Chitosan is an excellent biomaterial, whose nanoparticles have proved to be useful nanovehicle for carrying drugs, proteins, enzymes, and other substances. These nanocarriers may present remarkable applications in medicine and pharmacy.

4. Conclusions

Chitosan nanoparticles were prepared by nanoprecipitation technique starting from aqueous solution of N-(methylsulfonic acid) chitosan or directly in aqueous acetic acid. Methanol was used as the nonsolvent diffusing phase. Results showed that these nanoparticles became smaller as the polymer concentration decreased and, regardless of the polymer concentration, there is a tendency in the mean size to slightly diminish with increases in the amount of nonsolvent during the precipitation. Nanoparticles prepared in acidic media are slightly larger than those precipitated from N-(methylsulfonic acid) chitosan (400 vs. 200 nm approximately). Replacement of methanol by water in the suspension medium resulted in a notorious increase in their size of the particles. On the other hand, very little additions of Tween-80 to the nonsolvent phase render smaller nanoparticles, with a mean-size value very similar (around 300 nm) regardless of the nonsolvent to solvent volume ratio.

In experiments conducted employing low or high ionic strength chitosan solutions and methanol as the nonsolvent phase, there were no differences in size between nanoparticles. It is known that the ionic strength does not affect the value of the Flory's interaction parameter of chitosan solutions and therefore, it does not affect the excluded volume interaction, giving rise to nanoparticles with similar particle size.

These chitosan nanoparticles have good association and loading efficiency values of a model substance showing their ability as a nanocarrier for drug delivery systems.

Acknowledgements

Authors are grateful to Silvia Andrade and Goreti Campos, from the Scanning Electron Microscopy Laboratory at Centro de Investigaciones Científicas de Yucatán, for their contribution to the SEM analyses. This work was financed by Consejo Nacional de Ciencia y Tecnología, Mexico (CONACYT-SALUD-2008-C01-87324 project). AGLA thanks CONACYT for her PhD grant.

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