Applications of Chitosan Nanoparticles in Drug Delivery

Abstract

We have reviewed the binding affinities of several antitumor drugs doxorubicin (Dox), N-(trifluoroacetyl) doxorubicin (FDox), tamoxifen (Tam), 4-hydroxytamoxifen (4-Hydroxytam), and endoxifen (Endox) with chitosan nanoparticles of different sizes (chitosan-15, chitosan-100, and chitosan-200 KD) in order to evaluate the efficacy of chitosan nanocarriers in drug delivery systems. Spectroscopic and molecular modeling studies showed the binding sites and the stability of drug–polymer complexes. Drug–chitosan complexation occurred via hydrophobic and hydrophilic contacts as well as H-bonding network. Chitosan-100 KD was the more effective drug carrier than the chitosan-15 and chitosan-200 KD.

Key words : Nanoparticles, Chitosan, Drug delivery, Polymer, Binding site, Stability, Spectroscopy, Modeling

1 Introduction

Biodegradable and biocompatible chitosan (Scheme 1) and its derivatives have received major attention for the delivery of ther- apeutic drugs, proteins, and antigens [1–3]. Chitosan is a natural polymer obtained by a partial deacetylation of chitin [4]. It is nontoxic, biocompatible, and biodegradable polysaccharide.

Chitosan nanoparticles have gained more attention as drug delivery carriers because of their better stability, low toxicity, simple and mild preparation method, and providing versatile routes of administration [4–7]. The deacetylated chitosan backbone of glucosamine units has a high density of charged amine groups, permitting strong electrostatic interactions with proteins and genes that carry an overall negative charge at neutral pH condi- tions [4, 5]. The fast-expanding research of the useful physico- chemical and biological properties of chitosan has led to the recognition of the cationic polysaccharide, as a natural polymer for drug delivery [8, 9]. The encapsulation of several drugs and proteins with chitosan nanoparticles is recently reported, and major hydrophilic and hydrophobic contacts as well as H-bonding were dominated in drug–polymer complexation [3, 10–13]. Therefore, a comparative study of drug encapsulation with chito- san of different sizes will be of a major interest in order to evalu- ate the efficacy of chitosan nanoparticles in drug delivery.

Among drugs used to evaluate the efficacy of chitosan nanoparticles as nanocarriers were doxorubicin and its analogue FDOX known as antitumor agents against several types of cancers such as acute leukemia, malignant lymphoma, and breast cancer [14–16]. Similarly, tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen known as breast cancer drugs were used in this evalua- tion [17–20].

We have reviewed here the important role of chitosan nanopar- ticles as drug delivery tools, using doxorubicin, N-(trifluoroacetyl) doxorubicin, tamoxifen, 4-hydroxytamoxifen, and endoxifen with chitosan of different sizes: chitosan-15, chitosan-100, and chito- san-200 KD. The analysis of the drug binding sites and the stability of drug–polymer complexes were compared here in order to evalu- ate the efficacy of chitosan nanoparticles in drug delivery systems.

2 Materials

1. Purified chitosans 15, 100, and 200 KD (90 % deacetylation) (Polysciences, Inc., Warrington, USA) and used as supplied.
2. Doxorubicin hydrochloride (Pharmacia/Farmitalia Carlo Erba, Italy) and N-(trifluoroacetyl) doxorubicin were synthe- sized according to the published methods [21, 22].
3. Tamoxifen and 4-hydroxytamoxifen (Sigma Chemical Company) were used as supplied.
4. Synthesis of endoxifen was conducted at the Chemical Synthesis Core Facility by Fauq et al. [23].
5. Since chitosan is not soluble in aqueous solution at neutral pH (soluble in 0.1 N acetic acid or HCl), an appropriate amount of chitosan was dissolved in acetate solution (pH 5.5–6.5). Drug solutions were prepared in ethanol/water (25/75 %) and diluted in acetate buffer.

3 Methods

3.1 FTIR Spectroscopic Measurements

3.2 Fluorescence Spectroscopy

Infrared spectra were recorded on an FTIR spectrometer (Impact 420 model, Digilab), equipped with deuterated triglycine sulfate (DTGS) detector and KBr beam splitter, using AgBr windows. The solution of drug was added dropwise to the chitosan solution with constant stirring to ensure the formation of homogeneous solution and to reach the target drug concentrations of 15, 30, and 60 μM with a final chitosan concentration of 30 μM. Spectra were collected after 2 h incubation of chitosan with drug solution at room temperature, using hydrated films. Interferograms were accumulated over the spectral range of 4,000–600 cm−1 with a nominal resolution of 2 cm−1 and 100 scans. The difference spectra [(chitosan solution + drug solution) − (chitosan solution)] were generated using free chitosan band around 902 cm−1, as standard. This band is related to chitosan ring stretching [24, 25] and does not show alterations upon drug complexation. When producing difference spectra, this band was adjusted to the baseline level, in order to normalize the difference spectra.

Fluorimetric experiments were carried out on a PerkinElmer LS55 spectrometer. Stock solution of drug (30 μΜ) in acetate (pH 5.5–6.5) was also prepared at 24 ± 1 °C. Various solutions of chitosan (1–200 μM) were prepared from the above stock solutions by successive dilutions at 24 ± 1 °C. Samples containing 0.06 ml of the above drug solution and various polymer solutions were mixed to obtain final chitosan concentrations ranging from 1 to 200 μΜ with constant drug content (30 μΜ). The fluorescence spectra were recorded at λex = 480 nm and λem from 500 to 750 nm. The intensity of the bands at 592 nm from doxorubicin and its analogue [26] and at 360 nm for tamoxifen and its metabolites [27] was used to calculate the binding constant (K) according to previous reports [28–33].

3.3 Molecular Modeling

where F0 is the initial fluorescence intensity and F is the fluores- cence intensities in the presence of quenching agent (or interacting molecule). K is the Stern–Volmer quenching constant, [Q] is the molar concentration of quencher, and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution of the total emission for an interaction with a hydrophobic quencher [34, 35]. The K will be calculated from F0/F = K[Q] + 1.

The docking studies were carried out with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, WA, http:// www.arguslab.com). The chitosan structure was obtained from a literature report [36], and the drug three-dimensional structures were generated from PM3 semiempirical calculations using Chem3D Ultra 11.0. The whole polymer was selected as a poten- tial binding site since no prior knowledge of such site was available in the literature. The docking runs were performed on the ArgusDock docking engine using regular precision with a maxi- mum of 150 candidate poses. The conformations were ranked using the Ascore scoring function, which estimates the free bind- ing energy. Upon location of the potential binding sites, the docked complex conformations were optimized using a steepest decent algorithm until convergence, with a maximum of 20 iterations. Chitosan donor groups within a distance of 3.5 Å [37] relative to the drug were involved in complex formation.

4 Notes

1. Infrared difference spectroscopy was often used to characterize the nature of drug–polymer interactions [11, 12]. The major spectral shifting for the chitosan amide I band at 1,633– 1,620 cm−1 (mainly C=O stretch) and amide II band at 1,540– 1,520 cm−1 (C–N stretching coupled with N–H bending modes) [24, 25] together with the intensity changes of these bands obtained from difference spectra [(chitosan + drug solu- tion) − (chitosan solution)] was used to analyze the nature of drug–chitosan bindings, and the results are shown in Figs. 1, 2, and 3. Similarly, the infrared spectral changes of the free chitosan in the region of 3,500–2,800 cm−1 were compared with those of the drug–polymer adducts in order to determine the drug binding to polymer OH and NH2 groups, as well as the presence of hydrophobic and hydrophilic contacts in drug– chitosan complexes (Fig. 4).
The major increase in the intensity of chitosan amide I at 1,633–1,620 cm−1 and amide II at 1,540–1,520 cm−1 in the difference spectra of the drug–polymer complexes was used as the marker for drug–polymer interaction via chitosan NH2, N–H, and C=O groups (hydrophilic contacts). The positive features located at 1,660–1,620 cm−1 in the difference spectra of chitosan-15, chitosan-100, and chitosan-200 KD com- plexes with Dox, FDox, Tam, 4-Hydroxytam, and endoxifen are related to the increase in intensity of chitosan amide I band due to major drug–polymer hydrophilic interactions (Figs. 1, 2, and 3; diffs, 60 μΜ) [11, 12].

The analysis of the infrared spectra of chitosan in the region of 3,500–2,800 cm−1 showed major shifting of poly- mer OH, N–H, and CH stretching modes (Fig. 4). The poly- mer OH stretching vibrations at 3,460, 3,427 (free ch-15), 3,423 (free ch-100), and 3,449, 3,390 cm−1 (free ch-200) showed major shifting and intensity changes in the spectra of Dox and FDox, Tam, 4-Hydroxytam, and endoxifen chitosan complexes (Fig. 4a–c). Similarly, the N–H stretching vibrations at 3,230 (free ch-15), 3,245 (free ch-100), and 3,239 cm−1 (free ch-200) exhibit shifting upon drug complexation (Fig. 4a–c). The spectral changes of the polymer OH and N–H stretching modes are due to the participation of chitosan OH and NH2 group in drug–polymer complexes (hydrophilic contacts) [11, 12].

Fig. 1 FTIR spectra and difference spectra [(chitosan solution +drug solution) − (chito- san solution)] in the region of 1,800–600 cm−1 for the free chitosan-15 and its drug complexes for Dox-ch-15, FDox-ch-15, tamoxifen-ch-15, 4-hydroxytamoxifen-ch-15, and endoxifen-ch-15 in aqueous solution at pH 5–6 with 60 μM drug and chitosan concentrations.

Fig. 2 FTIR spectra and difference spectra [(chitosan solution + drug solution) − (chitosan solution)] in the region of 1,800–600 cm−1 for the free chitosan-100 and its drug complexes for Dox-ch-100, FDox-ch-100, tamoxifen-ch-100, 4-hydroxytamoxifen-ch-100, and endoxifen-ch-100 in aqueous solution at pH 5–6 with 60 μM drug and chitosan concentrations.

Hydrophobic interactions were also characterized by the shifting of the chitosan symmetric and antisymmetric CH stretching vibrations observed at 2,979, 2,862 (free ch-15), 2,941, 2,887 (free ch-100), and 2,938, 2,879 (free ch-200) in the spectra of Dox, Tam, 4-Hydroxytam, and endoxifen com- plexes (Fig. 4a–c). The overall spectral changes observed in this region (3,500–2,800 cm−1) were attributed to the presence of both hydrophilic and hydrophobic contacts in the drug–chitosan complexes [11, 12].

Fig. 3 FTIR spectra and difference spectra [(chitosan solution + drug solution) − (chitosan solution)] in the region of 1,800–600 cm−1 for the free chitosan-200 and its drug complexes for Dox-ch-200, FDox-ch-200, tamoxifen-ch-200, 4-hydroxytamoxifen-ch-200, and endoxifen-ch-200 in aqueous solution at pH 5–6 with 60 μM drug and chitosan concentrations.

2. Fluorescence quenching has been used as a convenient technique for quantifying the binding affinities of drug–polymer complexes [11, 12]. Since chitosan is a weak fluorophore, the titrations of Dox, FDox, Tam, 4-Hydroxytam, and endoxifen were done against various polymer concentrations, using drug emission bands at 350–750 nm [26, 27]. When drug interacts with chito- san, fluorescence may change depending on the impact of such interaction on the drug conformation or via direct quenching effect.

Fig. 4 FTIR spectra in the region of 3,500–2,800 cm−1 (polymer N–H and CH2 stretching vibrations) of hydrated films (pH 5–6) for free chitosan-15 (a), chitosan-100 (b), and chitosan-200 KD (c) and their drug complexes obtained with 60 μM polymer and 60 μM drug concentrations.

Fig. 5 Fluorescence emission spectra of drug–chitosan systems in 10 mM acetate buffer (pH 5–6) at 25 °C presented for (a) Dox-ch-15 and FDox and (b) Tam, 4-Hydroxytam, and endoxifen with free Dox (30 μM), with chitosan-15 at 10–100 μM; inset: K values calculated by F0/(F0 − F) versus 1/[chitosan] for drug–chitosan-15 complexes.

Fig. 6 Fluorescence emission spectra of drug–chitosan systems in 10 mM acetate buffer (pH 5–6) at 25 °C presented for (a) Dox-ch-100 and FDox and (b) Tam, 4-Hydroxytam, and endoxifen with free Dox (30 μM), with chitosan-100 at 10–100 μM; inset: K values calculated by F0/(F0 − F) versus 1/[chitosan] for drug–chitosan-100 complexes.

Fig. 7 Fluorescence emission spectra of drug–chitosan systems in 10 mM acetate buffer (pH 5–6) at 25 °C presented for (a) Dox-ch-200 and FDox and (b) Tam, 4-Hydroxytam, and endoxifen with free Dox (30 μM), with chitosan-200 at 10–100 μM; inset: K values calculated by F0/(F0 − F) versus 1/[chitosan] for drug–chitosan-200 complexes.

The f value calculated from Eq. 7 represents the mole fraction of the accessible population of fluorophore to quencher. The f values were from 0.25 to 0.65 for these drug–chitosan complexes, indicating a large portion of fluorophore was exposed to quencher.The number of drug binding site on polymer (n) is calcu- lated from log [(F0 − F)/F] = logKS + n log [chitosan] for the static quenching [38–47]. The n values from the slope of the straight-line plot showed between 2.8 and 0.5 sites are occupied by drug on chitosan molecule (Fig. 8 and Table 1). The results showed some degree of cooperativity for drug–polymer interaction.

In order to verify the presence of static or dynamic quenching in drug–chitosan complexes, we have plotted F0/F against Q to estimate the quenching constant (KQ), and the results are shown in Fig. 9. The plot of F0/F versus Q is a straight line for drug–chi- tosan adducts, indicating that the quenching is mainly static in these drug–polymer complexes (Fig. 9). The quenching constant KQ was estimated according to the Stern–Volmer equation:
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration, and Ksv is the Stern–Volmer quenching constant [48, 49], which can be written as Ksv = kQt0, where kQ is the bimolecular quenching rate constant and t0 is the lifetime of the fluoro- phore in the absence of quencher about 1.1 ns for free Dox and FDox around neutral pH [26, 50]. The quenching con- stants (KQ) are 1.8 × 1019M−1/s for Dox-ch-15, 1.3 × 1018 M−1/s for Dox-ch-100, and 2.6 × 1017 M−1/s for Dox-ch-200; 1.5 × 1018 M−1/s for FDox-ch-15, 9.3 × 1017 M−1/s for FDox-ch-100, and 2.7 × 1017 M−1/s for FDox-ch-200; 3.2 × 1016 M−1/s for Tam-ch-15, 2.6 × 1015 M−1/s for much greater than the maximum collisional quenching con- stant (2.0 × 1010M−1/s), the static quenching is dominant in these drug–chitosan complexes [48].

Fig. 8 The plot of log (F0 − F)/F as a function of log (chitosan concentrations) for the number of drug binding sites on chitosan (n) for drug–polymer complexes.

Fig. 9 Stern–Volmer plots of fluorescence quenching constant (Kq) for the chitosan and its drug complexes at different chitosan concentrations.

Fig. 10 Best docked conformations of drug–chitosan complexes for Dox, FDox, Tam, Hydroxytam, and endoxi- fen bound to chitosan with free binding energies.

3. Molecular modeling was often used to predict the binding sites for drug–polymer complexes. The spectroscopic results were combined with docking experiments in which Dox, FDox, Tam, 4-Hydroxytam, and endoxifen molecules were docked to chitosan to determine the preferred binding sites on the poly- mer. The models of the docking for drug are shown in Fig. 10. The docking results showed that drugs are surrounded by sev- eral donor atoms of chitosan C–O, N–H, and NH2 groups on the surface with a free binding energy of −3.89 (Dox), −3.76 (FDox), −3.46 (Tam), −3.54 (4-Hydroxytam), and −3.47 kcal/mol (Fig. 10). It should be noted that FDox is located near chitosan C–O, N–H, and NH2 groups with a hydrogen bonding system between drug O-213 and chitosan N-19 atoms (2.922 Ǻ) (Fig. 10). As one can see, drugs are not sur- rounded by similar donor groups showing different binding modes in these drug–chitosan complexes (Fig. 10).

5 Concluding Remarks

The spectroscopic and docking studies are strong analytical meth- ods to determine the binding parameters and the binding sites of drug–chitosan complexes. Major hydrophilic contacts via chitosan charged NH2 groups and hydrophobic interactions as well as H-bonding are observed in the drug–chitosan complexes. The order of drug–polymer binding is ch-100 > ch-200 > ch-15. Chitosan is stronger carrier for tamoxifen, 4-hydroxytamoxifen, and endoxifen than for doxorubicin and N-(trifluoroacetyl) doxo- rubicin in vitro. However, the addition of more soluble polymer such as PEG to chitosan will increase chitosan solubility and enhances Afimoxifene drug binding affinity both in vitro and in vivo [51].