Chapter 3 Experiment Methods
3.3 Drug diffusion test
The diffusion coefficients of the solutes were measured under switching MF (magnetic strength of about 400 Oe measured by Gauss meter) in a diffusion diaphragm cell (side-by-side cell) (see Fig. 3.2). The solution in the donor side is 80 ml of isotonic phosphate buffer (PBS) (pH7.4) containing 200 ppm of the model drug (vitamin B12). The receptor compartment, separated by the ferrogel, was filled with 80 ml of PBS solution. The concentration of each compound in the receptor compartment was determined at λ=361 nm using a UV spectrophotometer. The permeation coefficient (P, cm2/min) was calculated according to the following equation for the diaphragm cell:
where Cd0 is the initial concentration of the permeant in the donor compartment; Cd
and Cr are indicative of the concentrations in the donor side and receptor side, respectively; D is the diffusion coefficient (cm2/min) [Singha, 1999; Yang, 2003;
Miyajima, 1999; Liu, 2006]; H is the partition coefficient,; A is the effective area of the ferrogel; δ is the thickness of the ferrogel; V are respectively the volumes of solution in the donor and receptor compartment (both are 80 ml). By plotting ln[Cd0/(Cd–Cr)]
versus time (t), the permeability coefficient (P) can be calculated from the slope of the line by Eq.(3.5). Each data point was obtained by averaging of at least three measurements.
Moreover, the dry weight (Wdry) of drug-free ferrogel was immersed in the release medium until equilibrium state and then the wet weight (Wwet) were recorded.
Subsequently, the ferrogel was immersed in 10ml of vitamin B12-containing medium.
Partition coefficient (H) was determined from the initial (C0) and equilibrium (Ce) concentrations of vitamin B12-containing mediums by Eq. (3.6) [Miyajima, 1999; Liu, 2006].
Fig. 3.2 Diaphragm cell for measuring the permeability coefficient 3.4 Characteristics Analysis
The Raman spectra were obtained using a backscattering geometry. The 632 nm of a He-Ne laser was focused through an Olympus microscope with a 100× lens to give a spot size of 1 µm. The spectra was obtained using a 60 seconds acquisition time and averaged over 3 accumulations. X-ray diffractometer (XRD, M18XHF, Mac Science, Tokyo, Japan) was used to identify the crystallographic phase of ferrosponges, at a scanning rate of 6o per min over a range of 2θ from 10o to 70o. Additionally, the magnetization of the ferrogel was measured with a vibrating-sample magnetometer (VSM, Oxford) at 298K and ±6000G applied magnetic field. The structure of ferrogel and morphologies of magnetic nanoparticles were examined
r
K = partition coefficient
Cd = concentration of donor side Cr = concentration of receptor side δ= thickness of membrane C1
Cr Cd
Donor Side Receptor Side
Cd Cr
using field emission scanning electron microscopy (FE-SEM, JEOL-6500, Japan) and transimission electron microscope (TEM, JEOL-2010, Japan).
Pyrene loading was studied in order to assess the temperature-dependent aggregation within microgels. A stock solution of 1 mM pyrene in absolute methanol was prepared, from which 2 µl was added to 3 ml of 100 mg F127-shell MNPs aqueous sample. The sample was then allowed to equilibrate for 24 h at a given temperature, and then excitation and emission spectra were recorded using spectrofluorophotometer (PL). There are five vibrational peaks in the pyrene emission spectra. The ratio of the intensities of the first (373 nm) to the third (384 nm) vibronic peak (I372/I385) in the emission spectra of the monomer pyrene was used to estimate the polarity of the pyrene microenvironment [Bromberg, 1999 & 2003].
BET analysis (Quantachrome, NOVA 2000, USA) was conducted using N2 gas absorption isotherms at 77K, and the pore size were calculated following the approach by Barrett, Joyner, and Halenda (BJH). The nanospheres were dried by 80oC in vacuum condition for 1 day before BET analysis, afterwards the sample was de-gassed for 150oC and 2 hours following BET analysis [Hu, 2008].
The chemical structure of the activated polymers was characterized by NMR.
Proton nuclear magnetic resonance spectroscopy (1H-NMR) spectra were used to confirm the sites and degrees of substitution. The samples were dissolved in CDCl3
and the spectra were recorded by NMR spectrometer (Bruker Avance-500) at 500 MHz for proton, equipped with a microprocessor-controlled gradient unit and an inverse-detection multinuclear BBI probe with an actively shielded z-gradient coil. For size characterization of nanocapsules, dynamic light scattering (DLS, zetasizer-3000HS, Malvern, UK) was used. Microscopy was performed using a transmission electron microscope (TEM, JEM-2010, JEOL, Japan) operating at 200 kV to reveal the microstructure of nanocapsules.
In addition, the porosity of the ferrogels was determined by measuring the true density and the bulk density [Yang, 2003]. To measure the true density, a freeze-dried ferrogels were placed in a vacuum oven and the weight of the sample was measured (M). Afterwards, the ferrogels were put into the cell chamber cup of a pyconometer (Micromeritics, 1305) to measure the true volume (Vt). The true density (ρt) was then
calculated according to Eq. (3.7). To measure the bulk density, ferrogels were vacuum dried and then the area was measured. The thickness of the ferrogels was measured ten times with a digital gauge meter (Mitutoyo IDF-112) to obtain the bulk volume (Vb).
The bulk density (ρb) was then calculated according to Eq. (3.8). The porosity (ε) of the ferrogels was calculated according to Eq. (3.9).
ρt = M/Vt (3.7) ρb = M/Vb (3.8)
1
/ −
=Vbulk Vtrue
ε (3.9)
3.5 Methylthiazol tetrazolium (MTT) assay [Chen, Mat. Sci. Eng. C-Bio. S., 2005]
Cell (smooth muscle cell) proliferation was assessed using a methylthiazol tetrazolium (MTT) assay, which measured mitochondrial dehydrogenase activity of viable cells spectrophotometrically. MTT reagent is a pale yellow substrate which produces a dark blue formazan product when it is incubated with viable cells. Smooth muscle cell (SMCs, TG/HA-VSMC smooth muscle cell, human normal aorta smooth muscle cell, BCRC number: 60293) were purchased from FIRDI and grown in culture medium containing 90% F-12K medium with modifications-Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate supplemented with 10 mM HEPES, 10mM TES, 0.05 mg/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite and 0.03mg/ml ECGS and 10% fetal bovine serum. 1 mg of F127-MNPs sterilized by hydrogen peroxide gas plasma system (STERRAD® 50 system, a Johnson & Johnson Company, USA) and then placed into the wells of 6-well culture plates in contact with 2ml of SMCs (2×105 cells/ml, 3 to 4 passages) and were incubated in 5% CO2 at 37°C for 72 hr.
After cell culturing for 72 hr, the viability of SMCs was determined by MTT assay.
0.5 ml MTT (5 mg/ ml PBS solution, Sigma, USA) solution and 2ml serum free
medium was added to each well (6-well culture plates). After 4 hr incubation at 37oC, 2 ml of DMSO (dimethyl sulfoxide) was added to dissolve the formazan crystals. The dissolvable solution was jogged homogeneously about 15 min by the shaker. The optical density of the formazan solution was read on an UV–VIS spectrophotometer at 570 nm. All experiments were repeated five times. Data from the MTT assays were analyzed by means of Student’s t-test. A P-value less than 0.05 were considered to be significant.
Chapter 4
Biodegradable Ferrogel (Gelatin)
4.1 Introduction
In recent years, stimuli-responsive polymers, which can be responsive to external stimuli, such as pH, temperature, and electric field, have attracted a great deal of interest due to their potential applications in controlled drug delivery [Zrínyi, 2000].
Gelatin is a widely used polymer in pharmaceutical products [Cortesi, 1998].
Furthermore, it is of special interest in controlled release applications because of their soft tissue biocompatibility, the ease with which the drugs are dispersed in the matrix, and the high degree of control achieved by selecting the physical and chemical properties of the polymer network.
Magnetic materials have been widely used in the field of biotechnology in bio-separation, artificial muscles and drug carriers [Neuberger, 2005; Pankhurst, 2003;
Rosengart, 2005]. Some researchers have reported that the drug carriers of magnetic gels applied in targeting [Rotariu, 2005]. However, to our best knowledge, it is less seen to use magnetic fields to control drug release rate. Therefore, the combination of the gelatin and magnetic particles is a potential research to prepare the stimuli-polymer which can be applied in controlled drugs release by magnetic field.
Environmental sensitive hydrogels (smart hydrogels) with controlled drug release have been received great attention in the field of medicine, pharmaceutics, and biomaterials science. These hydrogels provides advantages over conventional therapeutic dosage forms by having higher delivery efficiency, site-specific delivery, controlled dose, and elimination or reduction of harmful side effects to the patients. By these wide advantages of the hydrogels, a number of researches have been successfully proposed to integrate active drug molecules and host materials, where to manipulate drug release desirably. For example, through conventional bolus injection, drug concentrations at site of therapeutic actions were only a portion of the treatment period in the therapeutic window [Uhrich, 1999]. By contrast, drug delivery from the controlled polymeric systems could maintain drug concentrations within the therapeutic window for prolonged time.
Such smart hydrogels possess such ‘sensing’ properties which allow to change in swelling behaviors, permeability, and elasticity upon only minute alternations in the environmental conditions. Many physical and chemical stimuli have been applied to induce various responses in response to change in temperature [Zhang, 2004;
Eeckman, 2004; Gutowska, 1997; Chiu, 2005; Claude, 2004], pH [Etrych, 2001; Chen, 2004], glucose [Chu, 2004], electric field [Murdan, 2003; Sutani, 2001] and magnetic field [Zrínyi, 2000], for the smart hydrogels [Qiu, 2001] which administer drug release considerably and can be potentially used in extended field. So far, many kinds of magnetic sensitive hydrogels (ferrogels) have been developed and studied with regard to biomedical materials. These hydrogels were usually prepared by introducing magnetic nanoparticles into a polymer matrix, and a macroscopic change in the shapes of the resulting ferrogels in response to external magnetic stimuli can be easily manipulated, which permit these ferrogels to be employed as muscle-like soft linear actuators and drug delivery systems [Mitsumata, 1999; Xulu, 2000; Zrínyi, 1998]. For example, magnetic-field-sensitive gelatin microspheres were reported for pulsed release of insulin via an oscillating magnetic field [Saslawski, 1988; Lu, 2005]
and the release rate of insulin in the alginate microspheres with magnetic particles is much faster than that in absence of an external magnetic field. Although magnetic nanoparticles (MNPs) were widely used for magnetic resonance contrast enhancement, tissue repair, immunoassay, hyperthermia, drug targeting and delivery and in cell separation [Gupta, 2005; Neuberger,2005], to the best of our knowledge, there has been little investigation on drug delivery under a direct current (DC) magnetic field through the use of magnetic nanoparticles in the ferrogels. Drug delivery from the magnetic sensitive ferrogels can be triggered by a non-contact force (an external magnetic field), which is superior to the traditional stimuli response polymers, such as pH or temperature sensitive polymer. By this concept, a Magnetic Targeted Carriers (MTCs) has been designed which could adsorb pharmaceutical agents through application of an externally magnetic field for site-specific targeting and sustained release of drugs [Fricker, 2001]. In addition, according to our previous study, it was demonstrated that a direct current (DC) magnetic field can be used to manipulate the drug release behaviors from a smart magnetic hydrogel [Liu, 2006]
through an on-off switch of a magnetic field.
However, a number of drawbacks still exist to use the magnetic hydrogels for drug delivery systems. For instance, it could not display fast and outstanding magnetic sensitive behaviors to control drug release. Zhang et al. reported that macroporous temperature-sensitive hydrogels exhibited a tremendously faster response to the external temperature changes due to their unique macroporous structures [Zhang, 2004 & 2001]. In addition, pHEMA sponges were developed to achieve rapid and reliable delivery of bioactive substances for long-term implantable drug delivery devices [Dziubla, 2001], and plasmid DNA with a sustained release from polymeric scaffolds was investigated for tissue regeneration [Storrie, 2006]. Therefore, in this work, a magnetic-sensitive sponge hydrogels (ferrosponges) was developed in this study to overcome those above-mentioned problems. The resulting ferrosponge is able to absorb a large amount of water and shows fast recovery property. Furthermore, magnetic sensitive walls of ferrosponges constructed by MNPs can effectively reduce their wall permeability and decrease the drug release via a given magnetic field, as shown in Fig. 4.1. For this purpose, a magnetic sponge hydrogel composed of a biocompatible gelatin and magnetic nanoparticles (MNPs) is investigated in terms of the concentrations of magnetic nanoparticle and gelatin. The drug release behavior from this ferrosponge in response to a magnetic field is investigated.
Fig. 4.1 Schematic drawing of drug release from the magnetic-sensitive ferrosponges with and without applying external magnetic field
4.2 Gelatin Ferrogel
4.2.1 Fabrication of gelatin ferrogel
For the preparation of the magnetic hygrogels (or called ferrogel), the gelatin (15 wt %) was first dissolved in deionized water at 45oC to ensure that the gelatin can be fully dissolved. After that, 4 wt% Fe3O4 nanoparticles (from Alfa Aesar) including 0.03 wt % drugs (vitamin B12, from Sigma) and genipin (Challenge Bioproducts Co., Ltd., Taiwan) with different weight ratio were added to the above gelatin solution under stirring for 30 min at 40 oC and then incubated in 25 oC for 2 days. The genipin-cross-linked ferrogel were respectively designated as Ge0.06, Ge0.03, Ge0.01 and Ge0.003 by their different cross-link density. For example, Ge0.06 means genipin content is 0.06 wt%.
The swelling rate of the ferrogels was measured as prescribed in our previous study [Yang, 2003] and the switching “on” mode of a given magnetic fields (MF) is about 400 Oe. For drugs release test, the ferrogels containing vitamin B12 were first immersed in 20 ml of phosphate buffer (PBS) (pH7.4) and then UV-visible spectroscopy was used for the characterization of absorbance peaks at 361 nm to determine the vitamin B12 release concentration.
4.2.2 Characterzation of gelatin ferrogel
Fig. 4.2-(a) showed the gelatin gels containing vitamin B12 (no Fe3O4 particles) were cross-linked by genipin with various weight ratios. After the gelatin/genipin solution was incubated for 2 days, the color of the gels transfers from pink (the color of vitamin B12) to purple (lower cross-linked density) or dark blue (higher cross-linked density). The UV spectrum in Fig. 4.2-(b) also demonstrated the color change. The above results may suggest that the gelatin could react with a variety of the genipin concentration to display different colors and morphologies. The darker the color of the gels, the denser the porosity of the gels, and the porosity or pore size of the gelatin gels would influence the drugs release properties. In addition, it was observed that the Fe3O4 nanoparticles were fairly uniformly distributed in gelatin hydrogels as evidenced from the cross-sectional SEM image in Fig. 4.3-(a). Hence, it also proves that the gelatin solution is an excellent dispersing agent for Fe3O4 nanoparticles.
Fig. 4.2 (a) OM photos of the gelatin gels with different cross-linked densities and (b) UV spectroscopy analysis of different cross-linked gelatin hygrogels
The swelling properties of the magnetic hydrogels as a function of switching MF were illustrated in Fig. 4.3-(b). While switching “on” mode of a given MF, it was found that not only swelling rate decreased sharply but also de-swelling in the differential curve. However, it will restore back to original states while switching “off” mode. The sensitive properties may be attributed to the fact that the porosity or the pore size of the ferrogels would decrease in switching “on” mode. The mechanism of the “close”
configuration of ferrogels can be further illustrated in Fig. 4.4. While the MF switching in “on” mode, the Fe3O4 particles tend to aggregate together and this causes the porosity of the ferrogel to decrease. Therefore, a swelling rate was reduced and a decreased drugs release rate was induced.
300 350 400 450 500 550 600 650 0.0
Ge0.06 Ge0.03 Ge0.01 Ge0.003
Table 4.1 Cumulative drugs release of the ferrogels in “on” or “off” mode of a given magnetic field at 120 min
Ferrogel Ge0.003 Ge0.01 Ge0.03 Ge0.06
MF OFF 56.5 % 52.2% 49.9% 48.0%
MF ON 47.4 % 45.5% 44.4% 44.1%
OFF- ON 9.1 % 6.7% 5.5% 3.9%
Fig. 4.3 (a) SEM observation of Fe3O4 nanoparticles distributed in gelatin hydrogels and (b) sensitive swelling rate of the ferrogels dependent on switching MF
(a)
100 nm
-0.01 0.00 0.01 0.02 0.03 0.04 0.05
Ge0.03 De-swelling
Swelling rate(fraction/min)
0 50 100 150 200 250 300 350 off
Magnetic Field
Time (min)
on
(b)
Fig. 4.4 Mechanism of “close” configuration of the ferrogels due to the aggregation of Fe3O4 nanoparticles under “on” MF causes the porosity of the ferrogels to decrease (Interparticle magnetic force)
Fig. 4.5-(a) exhibits the “close” configuration of Ge0.003 ferrogel, the drugs release rate decreased in switching “on” mode. Moreover, Table 4.1 shows the vitamin B12 release conditions of different cross-linked density ferrogel in MF switching
“on” or “off” mode. It was observed that the lower cross-linked density of the ferrogels, the more distinct the magnetic sensitive properties (OFF-ON) (9.1%). The reason is that the ferrogels with a lower cross-linked density display the softer properties which could cause the porosity to be easily modified due to the more free movement of the gelatin hydrogels chains of magnetic nanopaticles. Furthermore, the Ge0.003 ferrogel displays a higher magnetization (Ms) (9.199 emu/g) than the Ge0.03 ferrogels (6.023 emu/g) as measured from the vibrating sample magnetometer (VSM) and shown in Fig.4.5-(b). Based on the above two reasons, it could be demonstrated that the lower cross-linked density ferrogels display more obvious magnetic sensitive properties.
Besides, the magnetic sensitive properties of the drugs release could be found in continuous switching “on-off” mode for a given MF, as shown in Fig. 4.6. The differential curve of drugs release rate showed that the drugs release decrease in
= Magnetic Nanoparticles = Magnetic force
50 100 150 200 250 300
switching “on” mode, and restore original states in switching “off” mode. These sensitive characterizations are similar with the swelling rate.
Fig. 4.5 (a) Drugs release rate profiles of the Ge0.003 ferrogels in MF switching
“on”or “off” mode (b) hysteresis loop analysis of the magnetic hydrogels using VSM
Fig. 4.6 Sensitive drugs release properties of the ferrogels dependent on switching“on-off” mode for a given MF
(b) (a)
-15000 -10000 -5000 0 5000 10000 15000
-8
4.3 Gelatin Ferrosponges 4.3.1 Fabrication of ferrosponges
The commercially available gelatin from bovine skin (type A, ~300 bloom), 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Hydrochloride (EDC) and model drug vitamin B12 were purchased from Sigma Chemical Co.. Iron (II) chloride (FeCl2) and Iron (III) Chloride (FeCl3) were obtained from Fluka and Riedel-deHaen, respectively, and used as received. Ammonia hydroxide (NH4OH) in the form of 33%
water solution was obtained from Riedel-deHaen. Phosphate buffered saline (PBS) was purched form Ultra Biotechnology Corporation.
Magnetic sponge hydrogels (ferrosponges) were fabricated by in-situ co-precipitation process, and iron oxide nanoparticles were deposited directly in the gelatin hydrogel. Briefly, gelatin was dissolved in the D.I. water for 2 hours at 40oC.
After gelatin was fully dissolved in the solution, appropriate amount of FeCl2 and FeCl3
was added to the gelatin solution to form the hybrid sols. (The molar ratio of FeCl2/FeCl3 was kept constant at 2:1, and the reagents used for synthesis was showed in Table 4.2) When completely dissolved, the hybrid sols were rapid cooled to 4 oC to gel the gelatin which was then immersed in a water solution of NH4OH to start the iron oxide formation process. Immediately, the gels became black, indicating that the iron oxide nanoparticles have been formed in the system. After the in-situ co-precipitation of iron oxide nanoparticles, the ferrosponge were washed by D.I.
water for several times to remove un-reacted NH4OH solution, and then the ferrosponge were subsequently kept in the freezing baths maintained at -80 oC for 1 day and finally lyophilized in a freeze-dryer for 3 days. Finally, the macroporous structures were formed and cured by 1-ethyl-3-[3-(dimethylammino) propyl]
carbodiimide (EDC) in the 9:1 acetone: water solution at 4 oC.
Table 4.2 Reagents used for the synthesis of ferrosponges
aS%: Swelling ratio=(Weight of wet ferrosponge)/(Weight of dried ferrosponge)
bMNPs (wt%): The weight fraction of magnetic nanoparticles(MNPs) measured by TGA
4.3.2 Characterization of magnetic-sensitive ferrosponges
Magnetic-sensitive ferrosponges composed of gelatin and magnetic nanoparticles (MNPs) were prepared through an in-situ co-precipitation process. The traditional method of preparing iron oxide nanoparticles usually used the chemical co-precipitation of iron salts in the alkaline medium:
Fe2+ + 2Fe3+ +8OH− Fe(OH)2 + 2Fe(OH)3 → Fe3O4 + H2O
However, the iron oxide nanoparticles formed by using this traditional method aggregated easily [Lin, 2005]. To prevent aggregation, the gelatin and iron salts were mixed in advance to become a homogeneous mixture in which iron cation and the carboxylic acid groups of polymer allow to form a homogeneous complex structure in the solution [Lin, 2005]. While the ammonia solution was added, the iron oxide nanoparticles were directly formed in the presence of the gelatin, resulting in a sponge-like structure after lyophilizing. The ferrosponges shown in Fig. 4.7 exhibited a three-dimensional porous structure with macroporous and an anastomosing network of gelatin matrix. The mean pore size shown in Fig. 4.7-(a) for the 5 wt% gelatin matrix of ferrosponges was microscopically measured to be 100±23 µm, which was larger than that (50±24 µm) for the 15 wt% gelatin matrix of ferrosponges, Fig. 4.7-(b). It was found that the morphology and pore size of the ferrosponges seemed to be greatly dependent upon the gelatin matrix rather than the amounts of MNPs. The macropores
However, the iron oxide nanoparticles formed by using this traditional method aggregated easily [Lin, 2005]. To prevent aggregation, the gelatin and iron salts were mixed in advance to become a homogeneous mixture in which iron cation and the carboxylic acid groups of polymer allow to form a homogeneous complex structure in the solution [Lin, 2005]. While the ammonia solution was added, the iron oxide nanoparticles were directly formed in the presence of the gelatin, resulting in a sponge-like structure after lyophilizing. The ferrosponges shown in Fig. 4.7 exhibited a three-dimensional porous structure with macroporous and an anastomosing network of gelatin matrix. The mean pore size shown in Fig. 4.7-(a) for the 5 wt% gelatin matrix of ferrosponges was microscopically measured to be 100±23 µm, which was larger than that (50±24 µm) for the 15 wt% gelatin matrix of ferrosponges, Fig. 4.7-(b). It was found that the morphology and pore size of the ferrosponges seemed to be greatly dependent upon the gelatin matrix rather than the amounts of MNPs. The macropores