Chapter 2. Literature Review and Theory
2.2 Inorganic clay-organic (chitosan) hybrid composites
Over the last decade, the utility of inorganic nanoparticles as additives to enhance polymer performance has been established and now provides numerous commercial opportunities, ranging from advanced aerospace systems to commodity plastics. Low-volume additions (1-5 wt.-%) of highly anisotropic nanoparticles, such as layered silicates or carbon nanotubes, provide property enhancements with respect to the neat polymer that are comparable to those achieved by conventional loadings (1-40 wt.-%) of traditional fillers. In addition, unique value-added properties not normally
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possible with traditional fillers are also observed, such as reduced permeability, tailored biodegradability, optical clarity, self-passivation, electrical conductivity, electrostatic discharge, remote-actuated shape recovery, and flammability, oxidation, and ablation resistance. Although much attention gas been paid to polymer/clay nanocomposites, relatively little attention has been paid to biopolymer/clay nanocomposites. These are the cases of polylactide/clay nanocomposites [29], cotton/clay nanocomposites [30], poly(butylenes succinate)/clay nanocomposites [31] and plant oils/clay nanocomposites [32].
Chitosan has been extensively investigated for several decades for molecular separation, food packaging film, artificial skin, bone substitutes, water engineering and so on owing to its good biocompatibility, biodegradability, as well as multiple functional groups. However, its properties, such as thermal stability, hardness and gas barrier properties are frequently not good enough to meet those wide ranges of applications. Up to now, there is only a limited number of reports about the enhancement of properties of chitosan using polymer-layer silicate nanocomposite (PLSN) technology [33].
Asira had a preliminary study about chitosan-clay nanocomposites and reported a markedly improved tensile property but inferior thermal property of composites to that of pure chitosan [34]. Ruiz-Hitzky and his coworkers synthesized functional chitosan/mintmorillonite nanocomposites, which can effectively act as active phase for an electrochemical sensor in the detection of different anions [33]. Wang et al. [35] successfully synthesized chitosan/montmorillonite nanocomposites and reported that nanodispersed clay improved the thermal stability and enhanced the hardness and elastic modulus of the matrix systematically with increased clay loading, up to a loading of 10 wt.%. Because of the polycationic nature of chitosan in acidic
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media, this biopolymer also appears as an excellent candidate for intercalation in Na+-montmorillonite by mwans of cationic exchange processes [36]. On the other hand, an acidic pH value is necessary to provide –NH3+ groups in the chitosan structure. In such conditions, the adsorption process is mainly controlled by a cationic exchange mechanism due to the Coulombic interactions between the positive –NH3+ groups of the chitosan and the negative sites in the clay structure.
2.3 Self-assembles of modified chitosan derivatives
Chitosan usually has high molecular weight and strong network of intermolecular or intramolecular hydrogen bonds. Its poor solubility in water and common organic solvents has so far limited its widespread utilization. As a result, there have been many publications about the methods to enhance the solubility of chitosan, one of which was derivatization. For example, its solubility can be dramatically enhanced by introducing the carboxymethyl (CM) groups to the chitosan. The structure of CM-chitosan is similar to amino acids with amino group and carboxyl group in the molecule, and the difference from chitosan is the carboxymethyl group linked to the nitrogen or oxygen atom. Its good water-solubility, non-cytotoxicity and good bioactivity as functional biomaterial made CM-chitosan an important derivative [37]. Several biological properties of CM-chitosan [38] as well as its synthesis [39] have already been reported. Further, the aggregation behavior of CM-chitosan in neutral aqueous solution was investigated by Zhu et al. [40]. It was proposed that the driving force for the aggregation of CM-chitosan in the dilute solution is a combination of the effects of intermolecular H-bonding of CM-chitosan, electrostatic repulsions between COO- groups on the CM-chitosan chains and hydrophobic
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interaction among the hydrophobic moieties in CM-chitosan such as acetyl groups and glucosidic rings. Thus, CM-chitosan nanoparticle as carrier for the anticancer drug, doxorubicin (DOX), was evaluated by Du et al. [41]. It was found that the DOX release rate can be hindered by CM-chitosan nanoparticle with high molecular weight (MW) and degree of substitution (DS).
Polymeric amphiphiles consisting of hydrophilic and hydrophobic segments can form micelle or micelle-like self-assemblies with a hydrophobic core and a hydrophilic shell due to the intra- and/or intermolecular interactions of hydrophobic segments in aqueous media [42]. Hydrophobically modified chitosan derivatives such as alkylated chitosan [43] and deoxycholic-modified chitosan [44] have been focusing on recently due to their amphiphilic nature.
This novel kind of polymeric amphiphiles can form monodisperse self-aggregated nanoparticles in aqueous media, and their morphology can be controlled by the chemical structures of hydrophobically modified chitosan such as the molecular weight of chitosan, the types and the DS of hydrophobic groups [45]. Zhang et al. [46] reported the synthesis of Cholesterol-modified chitosan conjugate with succinyl linkages (CHCS). CHCS formed monodisperse nanoparticles in aqueous media and showed a potential as a sustained-release carrier of epirubicin in vitro. Delair et al. [47] reported the synthesis of biocompatible nanoparticles from the pH-induced self-complexation of the amphoteric polysaccharide N-sulfated chitosan.
These particles were assembled by electrostatic interactions between the protonated amino residues and the sulfate functions and stabilized by an excess of surface sulfate groups. Chung et al. [48] prepared the polymeric nanoparticles from amphiphilic chitosan derivatives fluorescenin isothiocyanate (FITC)-conjugated glycol chitosans that provided a novel and
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simple method for size control of self-assembled nanoparticles. Park et al. [49]
had prepared hydrophobically modified chitosan using linolenic acid as hydrophobic group. The hydrogel nanoparticles formed with hydrophobized chitosan was used as water soluble proteins such as BSA carriers. Fang et al.
[50] prepared the amphiphilic graft copolymer using chitosan as hydrophilic segment and poly (L-lactic acid) (PLLA) as hydrophobic segment through a protection-graft-deprotection route. The results indicated both hollow and solid spherical micelles were present in aqueous solutions.
However, due to the rigidity of the molecular chains in water, it is difficult for hydrophobically modified chitosan derivatives to form perfect spherical-shaped self-aggregated nanoparticles [51]. Hence, chitosan was hydrophilically modified by O-carboxymethylation to increase the flexibility of chitosan molecular chains in water, then followed by the hydrophobic modification with cholesterol to yield novel polymeric amphiphiles, cholesterol-modified O-carboxymethyl chitosan conjugates, which were used to prepare self-aggregated nanoparticles in water by probe sonication [52].
The results showed that the negatively charged carboxymethyl groups are advantageous for the formation of well-shaped and stable self-aggregated nanoparticles.
2.4 Chitosan nanoaggregates for drug delivery system
A general and widely accepted classification of nanoparticles has been given that the nanoparticles are solid colloidal particles with a diameter between 10 and 1000 nm. Among these colloidal particles, those formed by a shell-like wall with a liquid content may be considered as nanocapsules, while their solid counterparts are often referred to as nanospheres. Among the
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nanosized polymer materials, hollow polymeric nanospheres (nanocapsules) obtained particular interest because of their great potential in biomedical utilization, for instance, to encapsulate large quantities of therapeutic and diagnostic agents in their hollow cavities and release them at later stage. Such encapsulation can greatly increase drug bioavailability, protect agent from destructive factors upon parenteral administration, and modify their pharmacokinetics and biodistribution in body. Various methods, such as the self-assembly of block copolymers in selective solvent [53], layer-by-layer deposition of polyelectrolytes on temporary core [54] (which will be removed permanently at later stage), and microemulsion as well as microemulsion polymerization [55], has been developed to fabricate hollow polymeric spheres.
Jiang et al. [56] demonstrated a simple and direct method for fabricating hollow polymeric nanospheres with biocompatible and biodegrafable macromolecules.
In the approach, hollow polymeric nanospheres were formed in a completely aqueous system without the aid of surfactants, organic solvents, precursors of block and graft copolymers, template cores, or emulsion phase, and decreasing their potential toxicity. This hollow CS/poly(acrylic acid) (PAA) nanospheres as the drug carrier and their drug release pattern in vitro and in vivo with DOX as a model drug was further investigated [57]. The nanospheres showed a continuous release of the entrapped DOX up to 10 days in vitro and showed comparable in-vitro cytotoxicity against HepG2 cells compared to the free DOX.
Upon entrapment of therapeutic agents or drugs, oral administration is the most convenient and comfortable means of administering protein drugs and eliminates pain caused by an injection, the stress associated with multiple daily injections, and possible infections [58]. However, peptide drugs are
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poorly absorbed after oral administration because of their susceptibility to enzymatic degradation and their low permeability across the intestinal epithelium. Mucoadhesive polymers represent one class of biomaterials with an interesting potential for the design of trans-mucosal nanoparticulate carriers.
These polymers offer the possibility to facilitate the interaction of the nanocarrier with the intestinal mucosa and, hence, its access to the underlying epithelium. Indeed, this mechanistic principle has been adopted to explain the efficacy of particles made of acrylic polymers [59], polyanhydrides [60], and chitosan [61] as carriers for the trans-mucosal delivery of peptides. Alonso et al.
[] had shown that high molecular weight (MW>100kDa) chitosan nanocapsules are efficient vehicles for improving the oral absorption of salmon calcitonin.
Sung et al. [62] reported a simple ionic-gelation method to prepare nanoparticles composed of chitosan and poly(γ-glutamic acid) (γ-PGA) for oral insulin delivery. The in vivo results indicated that the insulin-loaded nanoparticles could effectively reduce the blood glucose level in a diabetic rat model. However, outside the range of pH 2.5-6.6, the nanoparticles became unstable and disintegrated. To overcome this problem, chitosan was conjugated with trimethyl groups for the synthesis of N-trimethyl chitosan (TMC) [63]. Nanoparticle self-assembled by the synthesized TMC and (γ-PGA) for oral delivery of insulin was successfully prepared and showed that TMC/γ-PGA nanoparticles were able to open the tight junctions between Caco-2 cells, and their effect on the tight junction’s integrity appeared to be reversible.
Chapter 3
Experiment Methods
3.1 Flowchart of Experiment Process
chitosan
Inorganic/organic hybrid Modification
Film nanoparticle Self-assembly
Electrical-stimuli release nanocapsule
Ratio of I/O Degree of hydrophobicity
Swell and Deswell behavior Drug release behavior Drug encapsulation
Surface potential Water state Particle size distribution
Fatigue property Morphology
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3.2 Materials
The chitosan used in this study was supplied by Aldrich-Sigma and used without purification. The same type of chitosan had been used by Darder et al.
who pointed out that the chitosan has an average molecular weight of 342500 g mole-1 and a deacetylation degree (DD) of ca. 75% [33].
As inorganic clay, Na-magadiite was purchased from Chang-Chun Petrochemical Co. (Hsinchu, Taiwan). The mean particle size and size distribution of the clay were measured by UPA (Ultrafine Particle Size Analyzer), Honeywell. The mean particle size of the clay (Na-magadiite) is estimated about 3 μm and a narrow particle size distribution of 2-4 μm can be observed.
Na+-Montmorillonite, supplied by Nanocor Co., is a Na+ form of layered smectite clay with a cationic exchange capacity (CEC) of 120 mequiv (100 g)–1. The MMT platelet shows a surface dimension of about 200-500nm in length and several ten nm in width.
Sodium phosphate for the preparation of buffers and acetic acid were purchased from Aldrich Chemicals. 2-propanol, sodium hydroxide, chloroacetic acid, acetic anhydride, hexanol anhydride, decanoic anhydride, and dodecanoic anhydride purchased from Sigma Co, USA, were reagent grade.
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3.3 Characteristics Analysis
The crystallographical structures of CS-clay hydrogel were determined using XRD diffractometer (XRD, M18XHF, Mac Science, Tokyo, Japan) equipped with a Cu Kα radiation source (λ=0.154 nm). The diffraction data were collected from 2θ= 1~30o at a scanning rate of 2o per minutes.
FTIR spectra were recorded with KBr pellets on a Bomem DA8.3 spectrometer (Canada) for tablet analysis in the spectral region (4000-400 cm-1) with 64 scans recorded at a resolution of 4 cm-1.
Malvern Zreasizer HS3000 photon correlation spectrometer with an applied voltage of 100V and a 5-cm quartz cell was used to determine the zeta potential values of clay (Na-magadiite) in the phosphate buffer solution (PBS) with different pH value (pH= 4, 6, 7, and 10). The concentration of clay in aqueous suspension was fixed at 0.1 wt%.
The tensile mechanical properties of the hybrid composites were measured using a complete system of MTS Tytron 250 and TestStar IIs system in the following conditions: crosshead speed, 10 mm min-1; test temperature, 25°C. The initial cross section (10 mm2) was used to calculate the tensile strengths and the tensile modulus. All results reflect the average of three measurements. The difference between each measurement is <5%.
The stability of the CS-MMT suspensions were characterized using the optical absorbance of a UV-VIS spectrometer (SP-8001, Metertech Inc.), at a wavelength of 550 nm. Square glass cuvettes with a path length of 1 cm were used as sample holder. All the data shown in the work is an average value of three measurements and the measurement error is well below 5%.
The mean size and size distribution of the nanocapsules were measured by dynamic light scattering (DLS) Nanoparticle Size Analyzer (LB-550,
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HORIBA, Japan). All measurements were done with a wavelength of 633.0 nm at 25oC with an angle detection of 90o. Each sample was repeatedly measured three times.
For elemental analyses, samples were extensively dried (80oC, 24h) prior to submission of samples. Elemental analyses were performed with a Heraeus Vario III-NCH elemental analyzer (Germany).
Morphological evaluation was performed by Transmission Electron Microscopy (TEM) (JEOL2100, Japan) and Scanning Electron Microscopy (SEM) (S6500, JEOL, Japan). Sample solutions were dropped onto the carbon-coated 300 mesh copper grids and dried at 50oC, then examined without being stained for TEM analysis. For SEM observation, sample was suspended into anhydrous ethanol, then dip-coated on the silicon substrate.
After evaporation at 50oC for 24 h, dried samples were coated with gold (~ 20 nm thickness) for analysis.
Proton nuclear magnetic resonance spectroscopy (1H-NMR) spectra were acquired to confirm the sites and degrees of substitution recorded by NMR spectrometer (Varian unityinova 500) at 270 MHz. The samples were dissolved at a concentration of 10 mg/ml in D2O and the spectra were performed at 353 K.
Differential scanning calorimeter (DSC, Perkin-Elmer instrument) was employed to identify the content and structural configuration of water molecules. The ACC solutions of 1.3% (w/v) were prepared by dissolving the obtained derivates in DI water. These suspensions were then cast onto petri dishes and dried at room temperature for 24 h, to form final dried samples.
Those dried samples were later subjecting to swelling in DI water with various time duration of 1, 2, 3, 4, and 5 min of swelling, respectively, corresponding to
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various amount of water content. Samples were quenched from room temperature to 213K and conditioned at 213 K for 10 min prior to the DSC test.
DSC curves were then obtained by heating the sample to 300 K at a scanning rate of 10 K min-1. The maximum content of non-freezable bound water (Wnf,max) can be determined by detecting the endothermic peak assigned to the first-order phase transformation of water in the samples with various contents of water. The endothermic peak of freezable bound water is not detected until a critical amount of water is added to the sample. The critical amount of water is correlated with the number of tight water binding sites.
Measurement of swelling behavior of CS-clay films
The film was first cut into round-shaped plate (its radius about 1 cm). The average thickness and the average weight of the obtained dried films were measured about 0.20 mm and 0.12 g, respectively. In order to measure the swelling ratio, each sample was weighed before and after immersion in phosphate buffer solution. After the excessive surface water had been removed with filter paper, the weight of swollen samples was measured at various time intervals. The procedure was repeated five times, until no further weight gain was detected. The swelling ratio was determined according to the following equation:
Swelling Ratio (%) = [(Ws-Wd)/Wd] × 100
where Ws and Wd represent the weight of swollen and dried samples, respectively. All results reflect the average of five measurements. The difference between each measurement is <5%.
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Electric-stimuli response behavior of CS-clay films
The film, pre-equilibrated and swollen in PBS, was cut into round-shaped plate (its radius about 1 cm) and weighed. Two platinum electrodes (its radius about 1.5 cm) were kept in contact with opposite surface of the film. The released water from hybrid films was continually removed using filter paper and the weight change of swollen hybrid films was checked periodically under electric field. The deswelling water ratio was evaluated as Wt/Wt0 where Wt0
and Wt were the initial weight of fully swollen films and the weight of films at deswelling time t, respectively. All results reflect the average of five measurements. The difference between each measurement is <5%.
Swelling under an applied voltage
The dry nanohydrogel, pre-equilibrated and swollen in 20 ml PBS (phosphate-buffered solution, pH7.4) for equilibrium, was cut into round-shaped plate (1 cm radius) and weighed. Two ring-shaped platinum electrodes (outer radius is 1.5 cm and inner radius is 0.5 cm) were kept in contact with opposite surface of the swollen nanohydrogel in the PBS. The electric voltage of 5V was applied from a dc power source for one hour. After the excessive surface water had been removed with filter paper, the weight of swollen samples was measured. The procedure was repeated three times, until no further weight gain was detected. All the data shown is an average of three measurements, where the measurement error is well below 5%.
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Drug Release under an applied voltage
2% vitamin B12 (relative to the total weight of the final suspensions) was added into those final suspension (prepared for forming nanohydrogel in the second stage.) prior to drying. The drug-loading nanohydrogel, pre-equilibrated and swollen in 20 ml PBS for five minutes, was cut into round-shaped plate (radius of 1 cm) and weighed. Two ring-shaped platinum electrodes (outer radius is 1.5 cm and inner radius is 0.5 cm) were kept in contact with opposite surface of the swollen nanohydrogel in the PBS. The electric voltage of 5V was applied from a dc power source [64]. At appropriate time intervals, 3 ml solution was extracted from the container and analyzed using a UV spectrophotometer (Agilent 8453) at a specific wavelength λ=361 nm.
For on-off switching operation, the drug-loading nanohydrogel swollen in 20 ml PBS was then kept in contact with two platinum electrodes for electric-stimulation. A repeated operation between switching on and switching off of the electric-stimuli of 5V were carried out for ten cycles and the time durations of switching between “on” and “off” are both five minutes. The amount of drug release was measured spectroscopically at various time intervals. Since there has a risk of measurement error of drug release rate between different batches of drug loads in the nanohydrogels upon repeated on-off operation, where the difference in drug concentration between each operation for different samples will cause a variation, mostly reduction, of the release rate for later stage of the on-off electric-stimuli release, we then defined a standard release rate (Rsd) by normalization of the release rate upon each cycle of test.
( )
[ M M M ] t
R
sd=
i−1−
i i−1 i≧1Here, Mi is the residual drug amount in the nanohydrogel for the ith electric-stimulation and t is the time of applied voltage of 5V (five min). The obtained values are an average of three measurements and the difference between each measurement is <5%.
Measurement of self-aggregation behavior
The pyrene solution (1.0 × 10-4 M) in methanol was added into the test tubes, and evaporated under a stream of nitrogen gas to remove the solvents.
Then, solutions of CHC self-aggregates in distilled water were, respectively, added into the above test tubes, brought the final concentration of pyrene to 1.0 × 10-6 M, which was nearly equal to the solubility of pyrene in water at 22oC [65]. The mixtures were sonicated for 30 min in an ultrasonic bath and shaken in a shaking air bath for 1 h at room temperature. Pyrene emission spectra were obtained using a fluorescence spectrophotometer (Hitachi FL-4500, Japan). The probe was excited at 343 nm, and the emission spectra were
Then, solutions of CHC self-aggregates in distilled water were, respectively, added into the above test tubes, brought the final concentration of pyrene to 1.0 × 10-6 M, which was nearly equal to the solubility of pyrene in water at 22oC [65]. The mixtures were sonicated for 30 min in an ultrasonic bath and shaken in a shaking air bath for 1 h at room temperature. Pyrene emission spectra were obtained using a fluorescence spectrophotometer (Hitachi FL-4500, Japan). The probe was excited at 343 nm, and the emission spectra were