Chitosan-based nanocomposites: Promising materials for drug delivery applications

CHAPTER

Chitosan-based nanocomposites: Promising materials for drug delivery applications

14 R. Onnainty, G. Granero

Department of Pharmaceutical Sciences, UNITEFA, CONICET, Faculty of Chemical Sciences, National University of Co´rdoba, Co´rdoba, Argentina

Chapter Outline 1 Introduction .......................................................................................................376 1.1 Inorganic compound: montmorillonite ....................................................376 1.2 Organic compound: chitosan (CS) .........................................................379 2 Bionanocomposites obtaining strategies ..............................................................380 2.1 Intercalation of the polymer in the mineral clay sheets ............................381 2.2 In situ intercalative polymerization ........................................................381 2.3 Melt intercalation ................................................................................381 2.4 Template synthesis ..............................................................................381 3 Characterization .................................................................................................382 3.1 X-Ray fluorescence (XRD) .....................................................................382 3.2 X-Ray diffraction (XRD) ........................................................................383 3.3 Infrared spectroscopy (IR) ....................................................................384 3.4 Thermal analysis: differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) ..........................................................385 3.5 Scanning electronic microscopy (SEM) ..................................................386 4 Toxicity studies ..................................................................................................387 5 Smectites-chitosan for drug delivery purposes .....................................................387 5.1 Determination of the in vitro drug release from bionancocomposites .........389 5.2 Mathematical analysis of drug release kinetics .......................................390 5.3 Mechanisms of controlling drug release from nanocomposite systems ..............................................................................................394 6 In vitro mucoadhesion determination of polymer-mineral clay nanocomposites ......396 6.1 Methods to determinate mucoadhesion ..................................................397

Biomedical Applications of Nanoparticles. https://doi.org/10.1016/B978-0-12-816506-5.00008-5 # 2019 Elsevier Inc. All rights reserved.

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7 Nanocomposite as bioadhesive-drug delivery systems for medical and pharmaceutical applications ...............................................................................401 8 Conclusions .......................................................................................................404 References ............................................................................................................404

1 Introduction In the pharmaceutical area, “nanocomposites” formed by the combination between natural polymers and mineral clays have attracted the attention of many researchers (Aguzzi et al., 2007). These materials obtained by two or more solid phases are called composites. Most of these hybrid materials are prepared by combining a polymer and a solid inorganic compound. Within this group are the nanocomposites, named so because at least one of their components is found as dispersed particles whose size is in the order of nanometers. These complex materials have the characteristics of combining the properties of their organic and inorganic components, such as swelling, mechanical, thermal, and bioadhesion properties (G€ unister et al., 2007). A wide variety of polymers are used to obtain nanocomposites, such as polylactic acid (PLA); polycaprolactone (PCL); proteins; and polysaccharides, like as chitosan (CS), alginate (ALG), and starch. When these materials are combined with microfibrous clay minerals like silicates, sepiolite, and palygorskite, they can get very interesting characteristics. Thus, it is preferred to use natural polymers when these nanocomposites are used for medical applications or for packing food. Because they are recyclable and biodegradable, these materials are called “green composites” or “bionanocomposites,” especially when the inorganic component is a silicate (Darder and Ruiz-hitzky, 2007). Particularly in pharmaceutical applications, hybrid materials obtained by the combination of clay minerals and biopolymers have aroused much interest (Viseras et al., 2010), especially when they are used for antibacterial coatings for medical instruments and wound dressings, for drug delivery systems, or for improving optical properties in the field of medical imaging (Besinis et al., 2015).

1.1 Inorganic compound: montmorillonite The structural characteristics of the mineral clay determine its physicochemical properties, being these very different within the group of clays as kaolins (1:1 phyllosilicates), smectites (2:1 phyllosilicates), and sepiolite (2:1 inverted ribbons), although they have the common feature of having a structure of octahedron and tetrahedron sheets (Maisanaba et al., 2015). The primary structure of a block of the mineral clay is a sheet formed by SiO4 4
tetrahedral and Al3+ or Mg2+ octahedral (Fig. 1). Mineral clays formed by Al3+, Mg2+, or Fe2+/3+ octahedral sheets, sandwiched between two Si tetrahedral sheets, are called smectites and belong to group of 2:1 clay minerals. These octahedron and also tetrahedron sheets have a negative charge,

1 Introduction

Al, Fe Mg, Li OH Tetrahedral

O Na, Mg

Octahedral

Tetrahedral

Exchangeable cations

FIG. 1 Schematic representation smectite clays.

which is compensated for interchangeable cations located in the space between the sheets of the clay, which produces isomorphic substitutions. On the other hand, 1:1 clay minerals formed by layers of a Si tetrahedral sheet and an Al octahedral sheet (kaolin group) or an Mg octahedral sheet belong to the serpentine group. They have no isomorph substitution and therefore no layer charge. There is a fundamental difference between 1:1 and 2:1 layers (Alexandre and Dubois, 2000). Solids most frequently used are laminar clays from smectite group, such as montmorillonite, hectorite, bentonite, or their derivative alkylammonium. Other mineral clays also used are lamellar double hydroxides (LDHs) (Parello et al., 2010; San Roma´n et al., 2012; Fernandes et al., 2014; Amaro et al., 2016), zirconium phosphate (α-ZrP) (Bhowmick et al., 2016, 2017), or lamellar perovskites (Liu et al., 2014).

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Montmorillonite (MMT) belongs to the smectite group and has a layered structure. Smectite-group clay minerals have large adsorption capacities for polymer molecules due to their special crystalline structure. MMT is a multilayered phyllosilicate clay formed by Si/Al oxide arranged in two tetrahedron sheets with an edge-bridged octahedral sheet (general structure 2:1 type), with a net negative charge. By isomorphic substitutions, the negative charge is neutralized by cations located in the space between the sheets of clay (mainly Na+ and Mg2+), which might be exchanged by a lot of organic molecules (Aguzzi et al., 2007). Silicate minerals have a layered structure, owning characteristics of good water absorption, swelling, adsorb ability, and cation-exchange ability, which are very important properties to produce pharmaceutical materials (Yuan et al., 2010). MMT and other mineral clays such as bentonite, when combined with natural polymers, form materials extremely useful for pharmaceutical and biomedical industries due to the properties they acquire and flexibility (Xie et al., 2013). In this regard, three different clay dispositions can be obtained (Fig. 2): (1)

(2)

(3)

Tactoid structures. Here, a true nanocomposite is not formed since the clay has little affinity for the polymer, and therefore, the expansion of the interlaminar space does not take place. Intercalated structures. Here, the intercalation of the polymer in the space between sheets is moderate, and therefore, it expands slightly with the entrance of the polymer chains to the interlaminar space of the clay, which determines that it retains its shape. Exfoliated structures. Here, the mineral clay loses its layered organization, separating into single sheets dispersed in the polymer matrix. In this case, the clay has a great affinity for the polymer (Maisanaba et al., 2015).

FIG. 2 Schematic representation of tactoid and intercalated structure of nanocomposites.

1 Introduction

1.2 Organic compound: chitosan (CS) Most of the research on bionanocomposites focuses on the use of materials like PLA, PCL, proteins, and polysaccharides, incorporating layered silicates of the smectite group. Of particular interest are polysaccharides like chitosan, derived from chitin, with positively charged amino groups; alginate, extracted from sea algae, with carboxylate negatively charged groups; and starch, obtained from maize, with a neutral charge (Alc^antara et al., 2014). Poly(lactic-co-glycolic acid) (PLGA) is widely used because of its biocompatibility, biodegradability, and versatile degradation kinetics with completely safe final degradation products (lactic and glycolic acids either are excreted by the kidneys or enter the Krebs cycle to be eventually eliminated as carbon dioxide and water); it has been most extensively used in designing drug delivery vehicles. It is also an FDAapproved biodegradable and biocompatible polymer that has been in use for years (Jain and Datta, 2014). Alginates are random, linear, and anionic polysaccharides consisting of linear copolymers of α-L-guluronate and β-D-mannuronate residues. Alginates have a long history of use in numerous biomedical applications, including drug delivery systems, as they are biodegradable, biocompatible, and mucoadhesive polymers. Alginate polymers are also hemocompatible and have not been found to accumulate in any major organs and show evidence of in vivo degradation. Sodium alginate (ALG) is used in a variety of oral and topical pharmaceutical formulations, and it has been specifically used for the aqueous microencapsulation of drugs, in contrast to more conventional solvent-based systems (Motwani et al., 2008). Chitosan (CS) is formed by β-(1,4)-linked 2-deoxy-2-amino-D-glucopyranose units, being the deacetylated product of chitin, poly(N-acetyl-D-glucopyranose) (Fig. 3). Cellulose is the second most plentiful natural biopolymer (Wang et al., 2005). It is a natural aminopolysaccharide. This polymer has a lot of biomedical applications and in other areas. Cellulose and CS are the most studied polymers worldwide because of their excellent biocompatibility and biodegradability

FIG. 3 Chemical structures of chitosan: D-glucopyranose units (X) and N-acetyl-Dglucopyranose (Y).

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properties, which make them ecologically safe, low toxic, and immunogenic materials (Pillai et al., 2009). Also, this polymer have a great affinity for many clays (Monvisade and Siriphannon, 2009). CS is considered a material with a big potential due to its versatility to allow its structural modification to obtain wishful properties and functional materials. Numerous research groups have reached a state of intense activity around the world, due to its positive attributions like excellent biocompatibility and biodegradability. Besides, CS has great mucoadhesive properties, whereby it is used to improve the time of permanence of drug transport systems that have it in their composition in the mucous membranes of the body (Ayensu et al., 2012). Polymers are usually filled with particles to enhance their properties, with special attention in its mechanical, stiffness, or toughness properties, or to reduce the cost of systems. Particles of nanometric size have more advantageous properties in relation to larger ones and, in particular, nanocomposites formed with clay minerals arouse great interest due to the natural abundance of their constituents (Hsu et al., 2012).

2 Bionanocomposites obtaining strategies The polymers interact with the mineral clay montmorillonite according to their ionic characteristics or nonionic character. When the polymer has ionic properties, it interacts electrostatically with the clay, while the polymers with neutral characteristics are adsorbed on the surface of the clay due to steric interactions. The concentration of the polymer, its molecular weight and their hydrolyzing groups, with the size and the shape of the clay particle, its surface charge, the clay concentration in suspension, pH, and temperature can affect the interactions that take place between the polymer and the mineral clay (G€ unister et al., 2007). Some strategies have been developed to synthetize nanocomposites. The different kinds of nanocomposites can be divided according to the Kormarneni’s classification: (I) Nanocomposites prepared by the low-temperature sol-gel method (II) Nanocomposites obtained by the polymer intercalation in the layer solid (III) Nanocomposites resulting from the polymer entrapment in the structure of the solid phase like zeolites (IV) Electroceramics, formed by ferroelectric, dielectric, and superconductor materials (V) Structural ceramic nanocomposites, obtained by the traditional methods at high temperatures (Komarneni, 1992) In this work, we will expose the most used four methods for producing polymerlayered silicate nanocomposites: in situ intercalative polymerization, intercalation of polymer or prepolymer from solution, melt intercalation, and template synthesis (Pavlidou and Papaspyrides, 2008; Alexandre and Dubois, 2000).

2 Bionanocomposites obtaining strategies

2.1 Intercalation of the polymer in the mineral clay sheets To obtain nanocomposites, first, the mineral clay must be exfoliated to produce simple sheets with a solvent in which the polymer is soluble or can be dispersed in the case of insoluble ones, such as a polyimide. Because the layers of the silicates are stacked by weak interactions, these clays can easily be dispersed in a suitable solvent. In a second stage, after the swelling of the clay in the solvent, the polymer is added to intercalate it in the clay. Finally, the solvent is removed, generally under vacuum by evaporation or by precipitation. After the solvent evaporation, the sheets of the clay are reassembled incorporating the polymer between their layers to obtain the nanocomposite. Under this process are also gathered nanocomposites obtained through the emulsion polymerization method, where the sheets of silicate are dispersed in the aqueous phase. This method has the advantage that nanocomposites can be synthesized using polymers with low or even no polarity. However, the disadvantage of this method is that a large volume of solvent is required, which makes its application at industrial scale quite difficult.

2.2 In situ intercalative polymerization Here, the layered silicate is swollen in the monomeric polymer solution, thus the polymer formation occurs between the sheets of the clay. The polymerization can be initiated before the swelling step, either by heat or radiation, or by the diffusion of a suitable initiator, or by catalyst through cationic exchange inside the interlayer.

2.3 Melt intercalation In this method, the silicate is mixed with the polymer, both in the molten state. When the surface of the clay is sufficiently compatible with the polymer, the latter can crawl into the interlayer space of the clay, forming either an intercalated or an exfoliated nanocomposite. The advantage of this technique is that it is not required to use a solvent.

2.4 Template synthesis This method is very used to obtain double-layer hydroxide-based nanocomposites. Here, silicates are formed in situ in a polymeric aqueous solution by self-assembly forces, where the polymer auspicious the nucleation and growth of the inorganic host crystals, being trapped between the sheets of clay. Among all exposed methods, the most used to obtain nanocomposites are the solgel and the polymer intercalation in layer solid techniques. In the latter, there are interactions of electrostatic type between both materials, like hydrogen bond and ion-dipole coordination (Darder and Ruiz-hitzky, 2007).

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3 Characterization To know whether added CS enters into the interlayer of the clay mineral or not, numerous techniques can be employed like as X-ray fluorescence (XRF), X-ray diffraction (XRD), infrared spectroscopy, differential scanning calorimetry (DSC)/thermogravimetric analysis (TGA), scanning electron microscope (SEM), and toxicological assays.

3.1 X-Ray fluorescence (XRD) In XRF elemental analysis, the sample is irradiated with high-energy X-rays. By the radiation, electrons are dislodged from the inner layers of the atoms, and the free positions they leave are immediately occupied by electrons from the outer layers. These outer electrons thus pass into a lower energy state, simultaneously emitting the released energy as X radiation of characteristic and typical wavelength of each kind of atom or element. The intensity of this emitted characteristic radiation, which is measured, is proportional to the quantities of the respective elements present in the sample being analyzed (Kn€ ofel, 1983). This technique is widely used for elemental and chemical analysis, particularly in the investigation of metals, glass, ceramics, building materials, etc. The XRF technique was used to determine the chemical composition of mineral clays. In this case, it presented the characteristics of MMT, as much as the clay in its pure state and the sodium clay, and both nanocompounds CS/MMT and CS/sodium montmorillonite (Na+ MMT), in order to compare the composition of each samples. The results obtained of the analyzed samples are shown in Table 1, where the chemical elements are presented in their oxide form. Table 1 XRF-Analysis of the MMT, Na+ MMT, CS/MMT, and CS/Na+ MMT Oxides/Sample

% Mass MMT

CS/MMT

Na+ MMT

CS/Na+ MMT

SiO2 AL2O3 Fe2O3 K2O MgO TiO2 CaO Br ZrO2 Na2O Cl C

77.9 14.2 3.61 2.06 1.29 0.693 0.194 0.0377 0.0356 – – –

69.5 14.3 3.33 1.76 1.51 0.576 0.0773 0.0086 0.0175 – – 8.94

71.3 13.4 3.37 1.97 1.94 0.767 – – 0.0386 7.23 – –

63.6 14.9 3.24 1.58 1.12 0.582 – – 0.0283 – – 15.00

3 Characterization

In the Na+ MMT sample, the presence of sodium (Na), aluminum (Al), and silicon (Si) could be clearly observed, while for the MMT sample in its original state, Na is absent. In this way, it is corroborated that different procedures used to intercalate Na+ in the structure of MMT were effective. Samples containing CS in their structure showed the presence of carbon (C), which is exclusively associated with the polymer present in the analyzed sample. Comparing the amount of C in each of the samples of CS/MMT and CS/Na+ MMT, 9 and 15% of CS were observed, respectively (Onnainty et al., 2016). This increase in the amount of CS, which is part of the Na+ MMT nanocomposite, could be due to the fact that Na+ ions are easily interchangeable with the polymer chains of CS (Monvisade and Siriphannon, 2009), favoring the interaction between the polymer and the clay, thereby increasing the proportion of the polymer in the system. X-ray fluorescence is a technique widely use with this kind of materials due the specific information that it provides about the sample.

3.2 X-Ray diffraction (XRD) XRD spectroscopy is a versatile and nondestructive technique that provides detailed information on the chemical and structural composition of the materials studied. Typical applications of this methodology include the determination of crystal structures, evaluation of polymorph and solvate structures, determination of crystallinity degree, and study of phase transitions. To know whether added CS enters into the interlayer of the clay mineral or not, XRD analyses were done to measure d-spacing of sodium montmorillonite (Na+ MMT). This value represents the thickness of the clay platelet plus the interlayer distance (Celis et al., 2012). The diffraction pattern of the Na+ MMT sample without CS presented a basal spacing peak at 2θ  9 degrees indicating that the thickness of the Na+ MMT silicate ˚ . CS showed typical pattern of a low crystalline powder, with diflayer was 12.5 A fraction bands around 2θ  10, 20, and 40 degrees. These low-intensity reflections are due to the crystalline regions formed by hydrogen bonds among the amino and hydroxyl groups on chitosan chains (Eloussaief et al., 2011). For the CS/ ˚ of the clay is slightly Na+ MMT nanocomposite, the basal diffraction at about 12.5 A shifted to a higher angle, that is, a smaller d-spacing (Onnainty et al., 2016). Thus, the observed decrease in the basal spacing indicates that Na+ MMT keeps the original crystal structure and exists as primary particles in the CS matrix, since the observed increase in the basal spacing can be considered as quite limited. Probably, due to the coiled structure of CS, its intercalation only occurs in the planar conformation, so that, although polymer molecules were effectively trapped inside the clay interlayer, part of them could remain at the outer surface of the clay particles (Celis et al., 2012; G€ unister et al., 2007). The diffraction patterns of the Na+ MMT in the range of 2θ  7–11 degrees presented the basal diffraction at 2θ  8.9 degrees, although the clay band moved slightly to the right, which would indicate that the CS did not enter enough into

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the interlaminar space of the mineral clay, but it would be present on the surface of the same (G€ unister et al., 2007). Because the Na+ MMT peak was maintained in all cases around 2θ  9 degrees, these results would indicate that the MMT interlayers remain occupied by the Na+ ions and not fully exchanged by the CS. Polymer would be adsorbed on the surface of the clay, due to the strong interactions that occur between the polymer and clay (Gao et al., 2014). On the other hand, the increase in the basal spacing observed in the diffractograms clearly suggests the intercalation of chitosan molecules between silicate layers. Due to the polycationic nature of chitosan at low acidic medium, the intercalation of chitosan molecules in the montmorillonite layers takes place mainly through a cationic exchange reaction. At the chitosan mixing ratio of 0.2, the basal ˚ (Dd ¼ 5.0 A ˚ ), indicating the parallel-type monolayer spacing increases to 14.6 A arrangement of interlayer chitosan molecules. When the chitosan mixing ratio (CMC) is increased, the basal spacing is proportionally increased. When the mixing ˚ (Dd ¼ 10.0 A ˚ ) suggesting the intercaratio is 10, the basal spacing reaches to 19.6 A lation of chitosan molecules as a bilayer conformation, corresponding to the thickness of two layers of chitosan chain plus the thickness of the acetate anions. The second chitosan layer is bonded to the first chitosan layer through hydrogen bonding interaction, since the anionic surface charge of the clay has been already balanced by the dNH3+ groups of the first chitosan layer. Thus, the dNH+3 groups of the second chitosan layer interact electrostatically with the acetate ions from the starting chitosan solution acting as available anionic exchange sites, as previously noted (Han et al., 2010). To summarize, this is a powerful technique that allows to characterize the nanocomposites about how the interaction is between the different compounds and the changes that the clay undergoes.

3.3 Infrared spectroscopy (IR) IR spectroscopy is a very useful technique for the characterization of solid, especially when the measurement is performed by the Fourier transform infrared spectroscopy (FT-IR) method. Infrared energy is a small portion within the electromagnetic spectrum and is divided into three regions, IR-distant (50–400 cm
1), IR-medium (400–4000 cm
1), and IR-near (4000–14,000 cm
1). This technique is used to obtain structural information, since it is based on the modes of vibration of a molecule. When the IR energy source irradiates a sample, the energy absorption by the sample is the result of transitions between the molecular levels of vibrational and rotational energy, these transitions being very sensitive to the details of the molecular structure. This produces structurally singular spectra useful for the identification of drugs and other organic substances. The FT-IR spectra of the nanocomposites and their pure components were compared and examined to determine possible interactions between them. The Na+ MMT spectrum showed a wide absorption band between 3100 and 3700 cm
1 due to the hydroxyl groups of the clay matrix and adsorbed water and

3 Characterization

an intense absorption band in the range 1000–1200 cm
1 due to the SidOdSi stretching of the silicate. Also, the characteristic band at 1632 cm
1 ascribed to the water molecules directly coordinated with the interchangeable cations of the clay (Hua et al., 2010; Aguzzi et al., 2014). The CS spectrum exhibited the peak associated to the vibration of carbonyl bonds of the amide groups (CONH-R) at 1656 cm
1 and a peak at 1587 cm
1 corresponding to the vibrations of the amine groups (NH2) (Fong et al., 2010). In addition, its spectrum showed a typical band between 3100 and 3700 cm
1 corresponding to the dOH and dNH groups (Paluszkiewicz et al., 2011). The peak at 1587 cm
1 of the dNH2 group in the starting CS was not observed in the CS/Na+ MMT nanocomposite spectrum, whereas a band appeared at 1514 cm
1 corresponding to the deformation vibration of the protonated amine group (dNH+3 ) of chitosan was displayed, indicating an electrostatic interaction between the cationic polymer and the negatively charged clay groups. In the FT-IR spectrum of the CS/ Na+ MMT nanocomposite, the CS peak corresponding to the dNH2 groups, initially at 1587 cm
1, had a shift to 1514 cm
1, which is associated with the vibrational deformation of the protonated amino group (dNH+3 ) of the CHI, indicating electrostatic interaction between the cationic polymer and the negative charges of the clay sialic groups (Monvisade and Siriphannon, 2009). In conclusion, FT-IR is useful to determine the interactions between the materials that form part of the new nanocomposite.

3.4 Thermal analysis: differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) Thermal analysis comprises a series of techniques that allow to determine changes in physical and/or chemical properties of a substance or system as a function of temperature, while subjecting the sample to a programmed and controlled temperature regime. In the study of the effect of temperature on solids, the use of combined techniques is advantageous. The combination of the DSC analysis with the TGA determination is very useful in the assignment of the observed thermal events. TGA and DSC represent the first-order analytic instruments for the accurate physical-chemical solid-state characterization of the components under study. Thermal analyses are commonly used as a routine method for rapid preliminary qualitative research. This method is based on comparing the thermal behavior of the pure components, their physical mixtures, and the new material synthetized by a standardized procedure (Giordano et al., 2001). The formation of the nanocomposite between Na+ MMT and CS generated numerous decomposition steps. The DSC curves of the CS/Na+ MMT system showed peaks at 87, 240, 457, and 563°C. The first event was associated with the dehydration of the nanocomposite, with a maximum at 87°C. Subsequently, comparing TGA curves of Na+ MMT, CS/Na+ MMT, and CS/ + Na MMT physical mixture (PM) (Fig. 4), it is possible to know the amount of CS in the nanocomposite and evaluate the stability of the polymer in this new

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FIG. 4 Comparative TGA graphics of CS, Na+ MMT, CS/Na+ MMT and CS/Na+ MMT physical mixtrure (PM).

material due to thr fact that in this experimental conditions, the CS decomposes completely and the Na+ MMT is stable. In this case, it was observed that CS/ Na+ MMT shows events of degradation and decomposition from the CS; these events occur with a maximum weight loss of 14%, while the water content of the nanocomposite was 2%. Analyzing the curves of CS/Na+ MMT and CS/Na+ MMT PM, it is possible to observe the CS stability increase when it forms the part of nanocomposite, because in the PM, it is observed clearly with the degradation patron of CS. The thermal analysis is a very useful technique to characterize this type of materials since it allows inferring so much amount of polymer that forms part of the nanocomposite.

3.5 Scanning electronic microscopy (SEM) The basis of the SEM is that the electrons emitted by a tungsten cathode pass through a column in which a high vacuum has been made. In it, the initial beam is concentrated by a series of electromagnetic lenses, that is to say, that its diameter decreases until becoming almost punctual. The punctual electronic beam travels over the entire surface of the sample as a brush that sweeps the sample continuously. This beam mobility is achieved thanks to a system of sweep coils located in the instrument column. In the interaction of the electron beam with the surface, secondary electrons are produced that, after being picked up by a detector, are placed on a scintillator, where each electron will give rise to several photons. These photons are directed to a photomultiplier through a series of diodes with increasing potential differences, and a large number of secondary electrons are produced by a cascade effect. What is achieved is

5 Smectites-chitosan for drug delivery purposes

an amplification of the current due to the original secondary electrons or, in other words, an amplification of the information on the supplied sample of said electrons. The secondary electrons, finally, prior passage through a video amplifier, are directed toward a tube similar to a cathode-ray oscilloscope on whose screen the image is produced. In summary, one of the main features of this instrument is the existence of a pointto-point correspondence established between the sample to be examined and the image formed. This correspondence is established at the same time, so that it covers the sample in time series, leaving the image divided into many photographic elements, which are captured by the system installed in the instrument and integrated into a single image that informs about the appearance of the material under study (Renau-piqueras and Faura, 1965). A SEM image obtained from a Na+ MMT sample was shown in Fig. 5A, where the laminar structure of the clay and the well-defined edges of this material could be identified. In the case of the CS/Na+ MMT sample, the image obtained by SEM (Fig. 5B) is different with respect to the clay image. The fundamental difference was that it was not possible to identify the laminar structure or well-defined edges observed in the image of the clay used as reference, since Na+ MMT would be uniformly coated by the CS (Onnainty et al., 2016). These images suggest that CS interacts effectively with clay, giving rise to an organic-inorganic hybrid nanocomposite, with a different morphology than its source materials.

4 Toxicity studies It is very difficult to reach a definitive and general conclusion about the toxicity or security of clays and their uses. It is important to highlight that the toxicity profiles of clay minerals and its derived nanocomposites are defined according to different parameters, including (I) the exposure conditions, such as the concentrations or exposure times assayed; (II) the experimental models selected; (III) the modifiers or surfactants incorporated in their structures and their concentrations; and (IV) the sensitivity of the assays performed. In conclusion, many benefits could be derived from clay minerals and their products, but a case-by-case toxicological evaluation is always required to avoid potential human and environmental risks (Maisanaba et al., 2015).

5 Smectites-chitosan for drug delivery purposes For therapeutic applications and with the objective of avoiding toxic or subtherapeutic concentrations, to reach therapeutic levels of the drug in the action site, during prolonged periods, to obtain the desired effect of the drug, are designed drug delivery

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FIG. 5 SEM images of (A) Na+ MMT Mag. 10 K x. (B) CS/Na+ MMT Mag 20 K x.

systems. Within these drug release systems are standout biopolymer/layered silicate material composites, also used for biomedical engineering strategies, due to their special structure and functional properties. Specifically, biopolymer-clay composites have the potential to develop critical formulations for biomedical purposes, tissue engineering strategies, and controlled

5 Smectites-chitosan for drug delivery purposes

drug delivery systems. The nanocomposites are formed by organic and inorganic solid interactions. These hybrid materials display outstanding properties; for example, they might be obtained with different biopolymers and several nanoscale particles, such as layered silicates (clay minerals). These hybrid materials can be manipulated to deliver and modulate the release kinetics of drugs transported in these systems. Composite have been obtained using several mineral clays like layered silicate mineral clays, for example, smectite clays (laponite, saponite, and montmorillonite). Within this group, MMT has advantageous properties like good adsorption and adhesive abilities, swelling capacity, and cation-exchange ability that make it an ideal material to be used in the preparation of drug release systems. Also, the FDA has approved this material for pharmaceutical use because it is considered a biocompatible material, for which reason it is used as an inert excipient in pharmaceutical products. However, a disadvantage for the preparation of controlled release systems is that the rate of release of the drug is sometimes too fast as a result of the very weak interactions between the drug and MMT (Kevadiya et al., 2010a,b). Natural polysaccharides, due to their nontoxicity, biocompatibility, and biodegradability, are widely being studied as biomaterial for drug delivery and tissue engineering applications. Within this group, the CS is found, which is characterized to be biocompatible, biodegradable, nontoxic, and a mucoadhesive polymer. However, its limited solubility in water and other organic solvents, in addition to its poor colloidal stability, limits its full exploitation in drug delivery systems. Also, due to its poor mechanical strength and high swelling ratio, CS leads to burst release of drugs by breaking down the 3-D network of the polymer (Dinu et al., 2016). To overcome these limitations, mineral-organic interactions can be used to sustain the release of active ingredients to improve their therapeutic utility, which may provide drug delivery systems with improved properties (Kevadiya et al., 2015).

5.1 Determination of the in vitro drug release from bionancocomposites 5.1.1 Dialysis bag technique Dialysis is a technique based on the diffusion of small solutes from a concentrated solution to a lower-concentration solution of this solute through a semipermeable membrane until equilibrium is reached, which is widely used in studies of in vitro drug release studies (Fig. 6). Particularly, the procedure to determine the in vitro velocity of the drug release from the nanocomposite systems consists of placing them in the form of dispersion in a buffer inside a dialysis bag, and at certain intervals, aliquots of the receptor medium in which the dialysis bag is submerged are taken, which are replaced by the same volume of fresh receptor medium preheated to 37°C, temperature at which these studies are generally performed, in order to keep the volume of the receptor medium constant.

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FIG. 6 Schematic representation of the dialysis bag technique for in vitro drug release studies.

5.1.2 Paddle method The release behaviors of nanocomposite powders could be conduce using the paddle method with an appropriate speed. Samples are immersed in the release medium maintaining at an adequate temperature (generally, 37°C). At specific time intervals, appropriate aliquots are removed, separated through a 0.45 μm microporous filter, to drug analytic analysis, and immediately replaced with an equal volume of release medium to keep the volume constant (Fig. 7).

5.1.3 Franz-diffusion cell It is suggested that in vitro drug release studies from nanocomposite systems using Frank diffusion cells employ a phosphate buffer and a dialysis membrane with a molecular weight cutoff (MWCO) of 10 kDa placed between the two compartments of the cell (Subramanian et al., 2014). The receiving compartment is maintained under agitation at 100 rpm with the help of a magnetic stirrer to ensure the homogeneity of the mixing, and at appropriate intervals, aliquots are taken from the receiving compartment, which are replaced with the same volume of fresh and preheated a 37°C (Fig. 8).

5.2 Mathematical analysis of drug release kinetics In order to understand the mechanism and kinetics of drug release, it is essential to fit the results of in vitro drug release study of well-known kinetic equations such as zero-order model (% cumulative drug release vs time), first-order model (log % cumulative drug remaining vs time), Higuchi model (% cumulative drug release vs square root of time), Hixson-Crowell model (cube root of % cumulative

5 Smectites-chitosan for drug delivery purposes

FIG. 7 Schematic representation of the paddle method equipment for in vitro drug release studies.

FIG. 8 Schematic representation of the horizontal Franz cell for in vitro drug release studies.

drug remaining vs time), and Korsmeyer-Peppas model equation (Dash et al., 2010; Mijowska et al., 2015).

5.2.1 Zero order release kinetics Drug release from the nanocomposite system when the drug release rate is independent from the drug concentration could be represented by the following equation:

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CHAPTER 14 Chitosan-based nanocomposites

Qo
Qt ¼ Ko t

(1)

Qt ¼ Q0 + K 0 t

(2)

Rearrangement of Eq. (1) yields: where Qt is the amount of drug release in time t, Q0 is the initial amount of drug in solution (most times, Q0 ¼ 0), and Ko is the zero-order release constant expressed in units of concentration/time. To study the release kinetics, data obtained from in vitro drug release studies are plotted as cumulative amount of drug release versus time. The value of K0 is obtained from the slope of the linear plot of cumulative % drug release versus time.

5.2.2 First-order model The first-order kinetics model describes the drug release from the system where release rate of the drug is concentration-dependent and can be expressed by the following equation: dC ¼
KC dt

(3)

where K is first-order rate constant expressed in units of time
1. Eq. (3) can be expressed as log Ct ¼ log C0
Kt =2:303

(4)

where Ct is the concentration of drug release in time t, C0 is the initial concentration of drug present in the nanocomposite system, Kt is the first-order rate constant, and t is the time. The data obtained are plotted as log cumulative percentage of drug remaining versus time that would yield a straight line with a slope of
Kt/2.303.

5.2.3 Higuchi model The Higuchi model describes drug release from an insoluble matrix as the square root of a time-dependent process based on Fickian diffusion. This model is based on the following hypotheses: (a) (b) (c) (d) (e)

Initial drug concentration in the matrix is much higher than drug solubility. Drug diffusion takes place only in one dimension (edge effect must be negligible). Drug particles are much smaller than system thickness. Drug diffusivity is constant. Perfect sink conditions are always attained in the release environment.

In general, the Higuchi model can be expressed as Qt ¼ KH xt0:5

(5)

where KH is the Higuchi constant. The value of KH is obtained from the slope of the linear plot of cumulative % drug release versus the square root of time.

5 Smectites-chitosan for drug delivery purposes

5.2.4 Hixson-crowell model This model assumes that the particle’s regular area is proportional to the cube root of its volume. The release of the drug from the nanocomposites that followed first-order kinetics can be expressed by the equation 1=3

Wo1=3
Wt

¼ kt

(6)

where Wo is the initial amount of drug in the formulation, Wt is the remaining amount of drug in the nanocomposite system at time t, and k (kappa) is a constant incorporating the surface-volume relation.

5.2.5 Korsmeyer-peppas model This model described the drug release from a polymeric system equation. To find out the mechanism of drug release, first 60% drug release data are fitted in KorsmeyerPeppas model according to the following equation: Mt =M∞ ¼ Ktn

(7)

where Mt/M∞ is the fraction of drug release at time t, K is release rate constant, and n is the release exponent. The logarithmic form of Korsmeyer-Peppas model is represented by the following equation: log Qt ¼ logK + nx log t

(8)

where n is the diffusion exponent, a measure of the primary mechanism of drug release. In this model, the value of n characterizes the release mechanism of drug as showed in Table 2. To find out the exponent of n, the portion of the release curve, where Mt/ M∞ < 0.6, should only be used. To study the release kinetics, data obtained from in vitro drug release studies are plotted as log cumulative percentage drug release versus log time.

Table 2 Interpretation of Diffusional Release Mechanisms From Polymeric Formulations Release Exponent (n)

Drug Transport Mechanism

Rate as a Function of Time

0.5 0.45 < n ¼ 0.89 0.89 Higher than 0.89

Fickian diffusion Non-Fickian transport Case II (relaxational) transport Super case II transport

t
0.5 Tn¼1 Zero-order release Tn¼1

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CHAPTER 14 Chitosan-based nanocomposites

5.3 Mechanisms of controlling drug release from nanocomposite systems 5.3.1 Burst release effect Some drug release profiles sometimes show a first stage of rapid release of the drug from their transporter systems, which is followed by a stable “plateau” profile. This first stage of short and rapid fast release of the drug is known as “burst release” (Fig. 9). Although in certain circumstances the rapid release of the drug from its formulation is a desirable process, in general, this type of drug release is unpredictable and undesirable, whose duration cannot be controlled, nor can the dose that is release be controlled, which could result in a toxic concentration of the drug, which is why this effect is treated to eliminate or minimize in most cases. The burst effect is attributed to the dissolution and diffusion of the drug trapped in the nanosystem near or on the surface of the nanocomposite. The next stage is characterized by the slower release of the drug, which is due to the diffusion of the drug attacked in the aqueous channels that form in the inner part of the polymer matrix of the nanocomposite. The burst phenomenon is usually observed with drugs that are very soluble in water and highly concentrated in the polymeric matrix. Nanda et al. (2011) showed an initial burst release of the anticancer drug paclitaxel from CS-polylactide (PLA)/MMT nanocomposites, which is attributed to a significant amount of paclitaxel initially associated with nanocomposites remained on their surfaces by weak interaction forces between CS-PLA/MMT and paclitaxel.

5.3.2 Mechanisms of release of the drug incorporated deeply into the polymer/mineral clay composite system Nanocomposites as drug delivery systems attempt to control drug concentrations in the target media with the aim of releasing the drug that transports in a constant way, during a prolonged period of time. To determine the mechanism that is involved in the drug release process, kinetic models are often used as zero-order,

FIG. 9 Burst release effect.

5 Smectites-chitosan for drug delivery purposes

first-order, Higuchi, Korsmeyer and Hixson-Crowell equations. The KorsmeyerPeppas model is also frequently used, which when used in polymeric release systems has the ability to establish whether the processes of swelling of the matrix (upon hydration) and gradual erosion of the matrix are involved in the phenomenon of the drug release (Dash et al., 2010).

5.3.2.1 Diffusion and swelling of the polymeric matrix Mineral clays when combined with a polymer have the ability to retard the release of the drug transported in the system and the erosion of the polymeric matrix due to the interactions that take place between the polymer and the mineral clay, particularly when the polymer is the chitosan (CS) and the MMT the mineral clay; these interactions take place in the amino groups of the CS that led to the reduction of chitosan swelling and, then, a decrease of the matrix erosion, being the swelling of the CS, which is the principal mechanism involved in the drug release. Also, the mineral clay creates tortuous pathways in the polymer matrix that retard diffusion of the drug into the system (Ambrogi et al., 2016). These phenomena have been also observed with nanocomposites of MMT with other polymers, that is, Dziadkowiec et al. (2016) loaded ibuprofen (IBU) into neutral guar gum (NGG)-montmorillonite and cationic guar gum (CGG)-montmorillonite, finding a reduced and sustained release of IBU from these nanocomposites, which was attributed to a reduced swelling of the polymer induced by well-ordered, intercalated structure of the clay-polymer nanocomposites. Intercalated structure, with CGG monolayer in the interlayer space, is likely to undergo less swelling in aqueous media than partially exfoliated structure as in case of IBU/MMT-NGG. Additionally, a relatively higher content of smectite clay also contributes to longer diffusion path length of the drug toward the dissolution medium and diffusion of the medium inside the clay-polymer nanocomposite itself, providing a specific tortuous path. Authors fitted drug release data to different kinetic models to assess the mechanism of IBU release from clay-polymer nanocomposites (zeroorder, first-order, and Higuchi release models). The best fit was obtained for the Higuchi release model due to the fact that IBU release rate was proportional to the square root of time (ft ¼ KH √ t, where ft is the amount of the drug dissolved, KH is the Higuchi dissolution constant, and t is the release time), which indicated that the release of IBU from nanocomposites may be attributed to the drug diffusion through the swollen polymer matrix. If the kinetics of drug release from the nanocomposite can be fitted to the model of Korsmeyer-Peppas (Mt/M∞ ¼ K tn), the values of n and K may be estimated. Onnainty et al. (2016) estimated n values <0.5 for chlorhexidine (CLX) loaded into CS and MMT nanocomposites. According to this n value, the CLX controlled release mechanism from the system was attributed to the diffusion of the drug through the swollen matrix with water-filled pores.

5.3.2.2 Erosion of the polymeric matrix The release of the drug from nanocomposite systems is produced not only by diffusion and swelling processes of the polymeric matrix but also sometimes by its erosion. Mijowska et al. (2015) fitted the release kinetic of methothrexate (MTX) from

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mesoporous silica nanoflakes by applying Higuchi and Korsmeyer-Peppas models and proposed a mechanism for the release of the drug to the concomitant processes of diffusion through water-filled mesopores and degradation of the mSiO2 matrix. Also, they estimated n values of 0.592 (37°C) and 0.773 (42°C), respectively, attributed to an anomalous diffusion (non-Fickian) of methotrexate (MTX) from mesoporous silica nanoflakes by fitting the first 60% of the MTX release data to the Korsmeyer-Peppas model, because the MTX release from the mSiO2 nanoparticles was controlled by more than one process. Nanda et al. (2011) by plotting the cumulative release data of paclitaxel (PTX) from CS and PLA at different ratios wt% of MMT versus time by fitting to an exponential equation of type of Mt/M∞ ¼ Ktn, showed a dependence on the drug loaded and the polymer content of the matrix. They obtained n values ranging from 0.55 to 1.68 by varying the amount of drug containing 5, 10, 15, 20, and 25 wt% and keeping chitosan (80%) and PLA (20%) constant, suggesting shifting of drug transport from non-Fickian to anomalous type, attributing the n value of >1 to a reduction in the regions of low microviscosity inside the matrix and closure of microcavities during the swollen state of the polymer.

6 In vitro mucoadhesion determination of polymer-mineral clay nanocomposites Mucoadhesive formulations are intended to be applied to the mucous membranes of the organism such as buccal, nasal, ocular, vaginal, pulmonary, and gastrointestinal mucosae. Bioadhesion or mucoadhesion is the binding of a substance, such as natural or modified natural hydrophilic polymers, to the biological tissue. A layer of mucus gel formed by water and a glycoprotein called mucins covers the mucous membranes of the body. Other components of the mucus are inorganic salts, carbohydrates, and lipids. Mucins have a protein core with branched oligosaccharide chains attached over 63% of its length, and they are those that give the mucus gel properties (Depan et al., 2014). Mucins are fibers linked together by noncovalent interactions such as hydrogen bonding and electrostatic interactions, which are completely hydrated, which is why they are a viscoelastic gel layer, and they are negatively charged due to the presence of anionic sialic, sulfate, and carboxyl functional groups (Ashton et al., 2013). To protect the mucous membranes of the body, many liters of mucus are secreted per day; thus, mucobioadhesive substances are promising materials to be used in the design of drug release systems to be administered in the mucous membranes of the body. As a strategy to transform a mucoadhesive release system is the use of positively charged polymer materials such as chitosan (CS), because this positively charged polymer can adhere to the mucus layer not only through electrostatic interactions but also by hydrogen bonds with mucins due to the presence of many amino groups in the polymer chains (Bravo-Osuna et al., 2007).

6 In vitro mucoadhesion determination

CS has been widely used to prepare nanocomposites with many different types of mineral clays. Mineral clays are characterized by improving thermal stability and mechanical properties of CS. Moreover, they retard the swelling properties of CS when intercalated nanocomposite structures are formed (Liu et al., 2007). This latter phenomenon is because the swelling degree of the polymer is reduced to the dehydration of the mucus gel to form adhesive joints, showing the polymers better mucoadhesion properties (Mortazavi and Smart, 1994). The improved properties of the mucoadhesion of the hydrogel of starch-graft-poly (methacrylic acid) were attributed to the incorporation of MMT in the hydrogel matrix (G€ uler et al., 2015).

6.1 Methods to determinate mucoadhesion Several techniques for in vitro determination of mucoadhesion have been reported in the literature. Some of them are discussed below.

6.1.1 TA-XT plus texture analyzer Many in vitro/ex vivo methodologies are based on the evaluation of mucoadhesive strength, that is, the force required to break the binding between the model membrane and the mucoadhesive formulation. Generally, the equipment used is a texture analyzer or a universal testing machine, such as the TA.XTplus Texture Analyzer. In this technique, the in vitro mucoadhesive properties of formulations may be assessed on porcine stomach tissue, bovine sublingual mucosa, bovine duodenal mucosa, gelatin or mucin disks, and mucin gel placed on the platform below the texture analyzer probe. Porcine mucosa is the membrane typically used for bioadhesive measurements. When the mucosa is held in ambient conditions without suspension in, for example, gastric fluid, a fixed volume of buffer is generally pipet onto the mucosa to standardize the hydration prior to testing (Fig. 10). Gelatin disks may be prepared by pouring 30% (w/w) aqueous solution of gelatin over a petri plate and left for jellification, while 10% mucin gel is absorbed on a cellulose fiber or mucin disks that are attached horizontally with double-sided adhesive tape to the lower end of a probe. Porcine mucin disks are prepared by compression in a Carver press for 30 s using a defined compression force (10 t) and horizontally attached to the bottom end of a TPA probe using sticky fixers. Immediately prior to mucoadhesive testing, the disk is hydrated by immersion in a 5% mucin solution for 30 s (Fig. 11). Samples of each formulation are packed into a shallow cylindrical vessel. The gelatin or mucin disk in the analytic probe is lowered onto the surface of the formulation applying a downward force for a predefined time; then, the probe is moved upward at a constant speed and measuring the force required to detach the gelatin or the mucin disk from the surface of the formulation (Fig. 12). The force required to detach the mucin disk from the surface of the formulation can be measured from a force-time plot. The maximum detachment force (Fmax) as a

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CHAPTER 14 Chitosan-based nanocomposites

FIG. 10 TA-XT Plus Texture analyzer for in vitro mucoadhesion determination using a mucosa membrane.

function of displacement is recorded, and the work of mucoadhesion (Wad), expressed in μL, is calculated from the area under the force versus distance curve (Fig. 13). Certain elements must be controlled to ensure maximum repeatability and consistency of results. These include the probe’s traveling speed, the amount and duration of applied force, and withdrawal distance and speed. G€ uler et al. (2015) evaluated the in vitro mucoadhesive properties of montmorillonite/starch-graft-poly(methacrylic acid) nanocomposite hydrogel (Mt/S-g-PMAA) using a TA-XT Plus Texture Analyzer and ewe vaginal mucosa as a model mucosa. In this study, the vaginal mucosa sample was placed on a bioadhesion test ring and hydrated with water. It was formed a tablet with the nanocomposite, which was attached to the lower end of the cylindrical probe with cyanoacrylate glue. Herein, work of adhesion (WA) (mJ/cm2) and maximal detachment force (MDF) (N) were calculated from force-distance plot.

6 In vitro mucoadhesion determination

FIG. 11 TA-XT Plus Texture analyzer for in vitro mucoadhesion determination using a mucin or gelatin disks.

FIG. 12 Process of mucoadhesive test by using a texture analyzer apparatus.

In this study, it was found that MMT has a synergistic effect on the mucoadhesive properties of the S-g-PMAA hydrogel due to a reduced swelling degree, required to prolong mucoadhesion, obtained by the incorporation of the mineral clay to the hydrogel matrix and formation of London-van der Waals forces and hydrogen bonding between MMT and mucin structure (Campbell et al., 2008). Salcedo et al. (2012) evaluated the mucoadhesive properties of a clay-polymer nanocomposite composed by MMT and CS by using a TA-XT2 Plus Texture

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CHAPTER 14 Chitosan-based nanocomposites

Force (N) 1

0.4000

2 Peak force

0.3000 0.2000

Work of adhesion

0.1000 0.0000 0.0

25.0

50.0

75.0

100.0

125.0

1.000

0.500

150.0 Time (s)

Debonding distance

0.000

−0.1000

FIG. 13 Force versus time plot.

Analyzer and porcine gastric mucin (PGM, type II). In this study, the nanocomposite sample was hydrated in pH 5.0 phosphate buffer and placed on a filter paper, which was fixed with double-sided adhesive tape on the bottom of the upper probe of the apparatus. The detachment force (mN) and the adhesive work (calculated from the area under the force-distance curve, AUC (mN*s)) were recorded. It was found that the MMT/CS nanocomposite had an intermediate mucoadhesive behavior between CS and MMT, where CS had the highest bioadhesive conduct.

6.1.2 Mucus glycoprotein assay Herein, the mucoadhesion is evaluated by incubating the drug formulation with mucin (i.e., from porcine stomach mucosa) and quantifying its adsorption onto their surfaces. The amount of mucin absorbed by the nanocomposite system is determined by subtracting the concentration of residual mucin in the suspension after adsorption from the total amount added (Fang et al., 2015).

6.1.3 Scanning electron microscopy (SEM) for mucoadhesion This method consists in putting a 0.1 mg/mL mucin solution in contact with the formulation in a PBS buffer pH 7.4 and allowing equilibrium to be reached by leaving them in contact for an adequate period of time. Subsequently, one drop of the

7 Bioadhesive-drug delivery systems

resulting mucin gel is dried in a vacuum and sputtered with gold before obtaining the SEM images, and changes in the SEM image of mucin fibers are attributed to mucoadhesion. Onnainty et al. (2016) observed the mucoadhesive properties of the chlorhexidine (CLX)-loaded sodium montmorillonite (Na+ MMT) and CS nanocomposite by using this technique. The SEM image (Fig. 14B) obtained when the pig gastric mucin (PGM) was put in contact with the CLX/CS:Na + MMT nanocomposite displayed a noticeable change in the mucin network in relation to the SEM image of the hydrated mucin free (Fig. 14A), in which mucin was observed as swollen and expanded fibers and, therefore, they occupy the entire volume of the mucin gel layer. In this case, the microstructural image of PGM in contact with the CLX nanocomposite system shows that PGM looks dehydrated, displaying a filamentous structure aggregated together and numerous pores with very small interfiber spacing. These noticeable changes in the mucin network after incubation with the CLX nanocomposite may be attributed to the water movement from the mucus gel to the contacting dry or partially hydrated formulation, leading to the dehydration of the mucus gel to form strong adhesive joints at low hydration (Xiang and Li, 2004; G€uler et al., 2015). Strong intermolecular and intramolecular electrostatic interactions take place between CS and mucin, mainly due to the fact that glycan’s mucins are terminated with sialic acid and sulfate groups, which can electrostatically interact with the positively charged CS from the CLX nanocomposite because the amino groups in the polymer chains are positively charged, which interacts electrostatically with the negatively charged sialic acid residues of mucin. Also, the probable formation of London-van der Waals forces and hydrogen bonding between MMT and mucin structure may contribute to the bioadhesive properties of the system.

6.1.4 Mucin particle method In this technique, mucoadhesive properties of the formulation are demonstrated by measuring the ZP changes of the mucin particles due to the presence of the test formulation.

7 Nanocomposite as bioadhesive-drug delivery systems for medical and pharmaceutical applications The new hybrid materials called nanocomposites obtained by the combination of polymers, like the biopolymer CS, and a mineral clay, like MMT, are very recently used systems for the formulation of drugs as a strategy for the controlled delivery of therapeutics due to the fact that not only these materials allow release of the drug molecules in the target place but also they are able to release the encapsulated or entrapped drug in slow and sustained manner, which makes it possible to reduce the toxicity of many therapeutic agents. Also, these systems are able to improve the absorption and therefore enhancing drug bioavailability by placing the drug delivery into mucosal surfaces.

401

402

SEM images of pig gastric mucin, PGM (A) and PGM incubated with the CLX/CS:Na+ MMT nanocomposite (B).

CHAPTER 14 Chitosan-based nanocomposites

FIG. 14

7 Bioadhesive-drug delivery systems

One of the limitations of the drug administration into the oral cavity is the important loss of drug by uncontrolled swallowing and salivary flow. A strategy to maintain therapeutic levels of the drug over an extended period of time in the oral mucosa is formulated drugs as nanocomposites (Aduba et al., 2013). Onnainty et al. (2016) obtained a CLX nanocomposite system by the combination of CS and MMT, with the aim of controlling the release of CLX into the mucosa of the oral cavity. CLX is a highly used antiseptic substance active against gram-positive and gram-negative bacteria, molds, yeasts, and viruses (Kolahi and Soolari, 2006). The obtained CLX nanocomposite showed good mucoadhesive and drug-controlled release properties. Among the advantages of mucoadhesive systems compared with traditional drug release systems are the following: 1. They are able to adhere to the mucous membranes of the body (oral, ocular, nasal, buccal, pulmonary, or vaginal) and increase the bioavailability of drugs. 2. They decrease the number of therapeutic doses needed by increasing the residence time of the pharmaceutical formulation in the administration site. 3. They can be applied in specific places of the body for the treatment of diseases such as sexually transmitted infections, inflammatory bowel disease, lung inflammation, and degenerative eye conditions. For example, several cancers such as colorectal cancer, among others, are treated with 5-fluorouracil (5-FU). However, this compound has short biological half-life and incomplete oral absorption. Also, this drug produces toxic side effects on the bone marrow and in the gastrointestinal tract (TGI), and it has a nonselective action against healthy cells (Li et al., 2008). To overcome all these disadvantages, Kevadiya et al. (2012) obtained 5-FU nanocomposites combining Na+ montmorillonite (Na+ MMT) and CS. 5-FU nanocomposites were able to reach therapeutic plasma drug concentrations after their oral administration in rats. Also, these 5-FU systems enhanced the residence time of the drug in the place of administration in comparison with the free 5-FU, reducing the drug toxicity and releasing the drug in a controlled way from obtained nanocomposites. 5-FU when was formulated as hybrid nanosystems was efficiently distributed to various tissues of the rat, showing a marked reduction of hepatotoxicity. Joshi et al. (2012) loaded the antiprotozoal agent quinine (QUI) in CS/MMT nanocomposite systems for colon-specific delivery and placed in gelatin capsules coated with Eudragit L 100 to prevent the drug release in the gastric environments after the oral administration to control the drug release. Nanocomposite systems obtained by the combination of CS and MMT were also applied for improving the intestinal permeability of drugs. Salcedo et al. (2014) obtained the oxytetracycline (OXT), a broad-spectrum antimicrobial agent, nanocomposite system by the combination of CS and MMT, which maintained the drug in vitro permeability across Caco-2 cell monolayers linear after a first stage of apparent decrease in the OXT permeability, unlike the free drug that showed an almost

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constant permeability due to the low intestinal permeability of OXT attributed to its efflux by P-glycoprotein (P-gp), while the nanocomposite could elude the P-gp efflux, resulting in an increasing drug permeability.

8 Conclusions Nanocomposites are new hybrid systems formed by the combination of polymers and inorganic materials, which have a promising utility in many areas, especially in medicine and for pharmaceutical applications because the materials to obtain them are very abundant in nature and inexpensive. In addition, when the polymer is of natural origin, such as chitosan, and the inorganic material is mineral clay, such as montmorillonite, these hybrid systems are characterized by being not toxic and biocompatible. In addition, when the polymer has mucoadhesive properties, these nanosystems are very powerful tools for designing formulations for drugcontrolled release.

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CHAPTER 14 Chitosan-based nanocomposites

Hua, S., et al., 2010. Controlled release of ofloxacin from chitosan-montmorillonite hydrogel. Appl. Clay Sci. 50 (1), 112–117. Jain, S., Datta, M., 2014. Montmorillonite-PLGA nanocomposites as an oral extended drug delivery vehicle for venlafaxine hydrochloride. Appl. Clay Sci. 99, 42–47. Joshi, G.V., et al., 2012. Confinement and controlled release of quinine on chitosanmontmorillonite bionanocomposites. J. Polym. Sci. A Polym. Chem. 50 (3), 423–430. Kevadiya, B.D., Joshi, G.V., Bajaj, H.C., 2010a. Layered bionanocomposites as carrier for procainamide. Int. J. Pharm. 388 (1–2), 280–286. Kevadiya, B.D., et al., 2010b. Montmorillonite-alginate nanocomposites as a drug delivery system: intercalation and in vitro release of vitamin B1 and vitamin B6. J. Biomater. Appl. 25 (2), 161–177. Kevadiya, B.D., et al., 2012. Layered inorganic nanocomposites: a promising carrier for 5fluorouracil (5-FU). Eur. J. Pharm. Biopharm. 81 (1), 91–101. Kevadiya, B.D., Rajkumar, S., Bajaj, H.C., 2015. Application and evaluation of layered silicate–chitosan composites for site specific delivery of diclofenac. Biocybernet. Biomed. Eng. 35 (2), 120–127. Kn€ofel, D., 1983. Quimica del Cemento. Calidad del cemento. In: Carrete, J.V.B.A. (Ed.), Prontuario del cemento. Reverte S.A., Barcelona, pp. 145–235. Kolahi, J., Soolari, A., 2006. Rinsing with chlorhexidine gluconate solution after brushing and flossing teeth: a systematic review of effectiveness. Quintessence Int. 37 (8), 605–612. Komarneni, S., 1992. Feature article nanocomposites. J. Mater. Chem. 2 (12), 1219–1230. Li, S., et al., 2008. Pharmacokinetic characteristics and anticancer effects of 5-fluorouracil loaded nanoparticles. BMC Cancer 8, 103. Liu, K.H., et al., 2007. Effect of clay content on electrostimulus deformation and volume recovery behavior of a clay-chitosan hybrid composite. Acta Biomater. 3 (6), 919–926. Liu, P., et al., 2014. Palygorskite/polystyrene nanocomposites via facile in-situ bulk polymerization: gelation and thermal properties. Appl. Clay Sci. 100 (C), 95–101. Maisanaba, S., et al., 2015. Toxicological evaluation of clay minerals and derived nanocomposites: a review. Environ. Res. 138, 233–254. Mijowska, E., et al., 2015. Sandwich-like mesoporous silica flakes for anticancer drug transport—synthesis, characterization and kinetics release study. Colloids Surf. B: Biointerfaces 136, 119–125. Monvisade, P., Siriphannon, P., 2009. Chitosan intercalated montmorillonite: preparation, characterization and cationic dye adsorption. Appl. Clay Sci. 42 (3–4), 427–431. Mortazavi, A.S., Smart, J.D., 1994. An in-vitro method for assessing the duration of mucoadhesion. J. Control. Release 31, 207–212. Motwani, S.K., et al., 2008. Chitosan-sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: formulation, optimisation and in vitro characterisation. Eur. J. Pharm. Biopharm. 68 (3), 513–525. Nanda, R., Sasmal, A., Nayak, P.L., 2011. Preparation and characterization of chitosanpolylactide composites blended with Cloisite 30B for control release of the anticancer drug paclitaxel. Carbohydr. Polym. 83 (2), 988–994. Onnainty, R., et al., 2016. Targeted chitosan-based bionanocomposites for controlled oral mucosal delivery of chlorhexidine. Int. J. Pharm. 509 (1–2), 408–418. Paluszkiewicz, C., et al., 2011. FT-IR study of montmorillonite-chitosan nanocomposite materials. Spectrochim. Acta A Mol. Biomol. Spectrosc. 79 (4), 784–788.

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