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Porosity and dielectric properties as tools to predict drug release trends from hydrogels
© Raja and Fathima; licensee Springer. 2014
Received: 30 April 2014
Accepted: 25 July 2014
Published: 29 July 2014
Conventional studies on hydrogel properties such as viscosity, pH and swelling provide information without treating the components of hydrogel, viz., water and polymer individually. Water and hydrophilic polymers need to be studied individually to understand their relationship with each other to relate their influence on drug release. In this context, we have assigned the combination of porosity and dielectric properties as tools to explore the hydrogels. Porosity and dielectric properties have been analyzed using thermoporometry and alternative current impedance measurements, respectively. A well-known hydrogel genipin cross linked gelatin-chitosan (GC) composite, with catechin as model drug has been studied. The increasing concentration of chitosan in the hydrogel composites led to increase in bound water content and incorporation of charge entrapping moieties. Controlled and medium drug release are observed for GC1 whereas the native hydrogels and composites with lower ratio of chitosan yield immediate release and composites with higher ratio effects in slow release for limited duration (9 hours) of drug delivery process. This trend of drug release is in accordance with the results obtained from porosity and dielectric properties where reduction in pore radii to lower range and increase in relaxation time of polymeric components were observed at higher concentration of chitosan. Thus, these properties can be judiciously used for predicting drug release and designing biomaterials according to it.
Hydrogels consist of hydrophilic polymers dispersed in water medium and hence termed as colloidal solution (Sarkhejiya and Baldaniya2012). The release of drug molecules from a hydrogel matrix is usually predicted using the properties such as swelling, viscosity, pH, biodegradation and degree of cross linking (Cheng et al.2014; Soni and Singhai et al.2013; Sun et al.2013; Zhang et al.2013). Though these properties emphasize the whole hydrogel system itself, they fail to characterize the individual components present in it. Porosity and dielectric properties bear advantage over other properties as the impact of water is highlighted for pore size determination and that of polymeric component is stressed for analyzing admittance and dielectric relaxation (Kanungo et al.2011;2013a).
Porosity measurement using thermoporometry gains advantage over other techniques such as Brunauer-Emmett-Teller (BET) and Mercury Intrusion Porosimetry (MIP), as the latter demands the sample to be in dried state (Chae et al.2013; Fathima et al.2002; Qu et al.2006). Thermoporometry determines pore size based on melting or crystallization point of water molecules confined into the pores of hydrogels. Analyzing thermodynamic behavior of water with respect to its interaction with polymers helps evaluate different types of water and hence provide a range of pore sizes in nano range (Landry2005; Yamamoto et al.2005a,[b]). Alternative Current (AC) impedance analysis is a promising tool to explore dielectric properties of bio materials. Hydrogels are present in human body in the form of mucus, cartilage and vitreous humor of eye (Dean et al.2008; Schaefer et al.2002) and are acting as dielectric materials (Ren and Lam2008).
Gelatin is a well-known protein and is a denatured form of collagen (Zeugolis and Rahunath2010). As breakdown products of gelatin are not harmful for human beings, they are known to be biocompatible (Pulieri et al.2008). Gelatin has been widely used for various types of biomedical applications such as contact lenses, wound dressing, food additives including drug delivery (Dongargaonkar et al.2013; Ngo et al.2014; Shi and Tan2004). Chitosan is a linear polysaccharide consisting of reactive amine and hydroxyl groups. Chitosan and its derivative have been proven as safe candidates for mucosal and trans- mucosal delivery of drugs (Cui et al.2014; Rodrigues et al.2012). Hence, in this present study, gelatin-chitosan composite hydrogels have been taken as model system. Though composites are preferred in many of the applications to enhance physical properties of native hydrogels in the aspect of elasticity, tensile strength and thermal stability (Parvez et al.2012; Yin et al.2010), the rationale behind selecting copolymer, in our work, is to enhance porosity attaining well cross linked network for better drug release profile. For the purpose of bringing about permanent cross linkage between polymers, genipin, a biological cross linking agent is taken during composite preparation. Genipin involves in covalent bond formation between free NH2 groups found in lysine and arginine of gelatin and present on each residue of chitosan. The pH value 7 and temperature above 40°C enhance the rate of reaction to form genipin dimer between polymers. The proposed formation structure of blue pigments (genipin dimer) from genipin is shown in previous reports (Chaubaroux et al.2012; Wang et al.2012).
Hydrogels are one of the most widely used drug delivery agents for various kinds of drugs such as anti-oxidant, hormone and protein (Park et al.2013; Peng et al.2012; Van Thienen2007). Catechin, a free radical scavenger containing hydroxyl groups is taken as model drug for in vitro study. It is reported that catechin accounts for low bioavailability when administered orally. It is metabolized into conjugated mainly methylated form, which is not active against oxidative stress (Baba et al.2001; Veluri et al.2004). When the anti-oxidant encapsulated into hydrogels scaffold is injected intravenously, the availability of active ingredients of catechin in blood can be increased.
The objective of this work is to elucidate the dispersion medium (water) and dispersed phase (polymers) of hydrogels through porosity and dielectric properties and to relate the effect of these properties with drug release profile.
Results and discussion
Porosity and dielectric properties of hydrogels
Genipin dimer formation was confirmed from the UV absorption peak at 602 nm. The composite GC3 shows maximum absorbance and is five times higher than the lower ratio of composites (GC0.3, GC0.5 and GC1) whereas the composite GC2 shows twice higher absorbance. Gelatin contains primary amino groups in amino acids such as lysine and arginine and participates in cross linkage with reactive amine groups of chitosan involving inter and intra cross linking up to particular percentage of copolymer blended. When chitosan percentage increases, they have the propensity to involve intra cross linkage within chitosan as availability of amino groups of gelatin is very less than that of chitosan. This leads to high level of cross linkage in polymeric network of hydrogel composites (GC2 and GC3).
Further, the free molecular motion in solution becomes less for GC2 and GC3 after 12 hours incubation. The mobility of polymeric components in these two composites is hindered significantly and that can be understood from the shift (indicated by perpendicular arrow) from base line observed in UV spectrum. This shows that genipin has strong impact in forming higher degree of cross linked network in composites GC2 and GC3 as the concentration of chitosan is increased. These cross linkage and blended polymers in composites have direct link with changes in dynamics of water present in hydrogels.
The thermodynamic parameters and amount of freezable water of hydrogels determined from DSC
Onset temperature (°C)
Amount of freezable water (%)
Where, ∆T is the difference between temperature at any point (Ti) and normal melting point (T0) of water. Ri and Ri+1 are pore radii corresponding to temperature at successive points of temperature. ∆V is pore volume (cm3/g) at a temperature interval.
As shown in Figure 3, the pore radii of nano sized pores in each hydrogel are measured. Though all the hydrogels are having pores in the range of 7 to 100 nm, GC2 and GC3 have pore radii in the range 7–50 and 7–40 nm, respectively, due to impact of genipin cross linkage with increasing concentration of chitosan. As can be seen in figure, the intensified peaks correspond to availability or number of pores with same radii. For GT, large numbers of pores are found within the range 40–55 nm. The gradual decrease in intensity of peaks is observed for CT indicating the numbers of pores with smaller radii are larger than that of higher radii. The pore size measured for CT, GC0.3, GC0.5 and GC1 were 7–100, 7–100, 7–80 and 7–60 nm, respectively. When chitosan is incorporated into gelatin, all the composites are showing more intensified peaks towards lower radii range. Hence, it is understood that chitosan has imparted more and reduced porous network for this gelatin based hydrogel system with the aid of genipin cross linkage. Though CT and GC0.3 are showing similar range of pore radii, the intensity of peaks of CT is more than GC0.3 at lower radii range reflecting the content of bound water measured through thermodynamic values.
Where, Z′(ω) and Z″(ω) are real and imaginary components of the total impedance, Z(ω), respectively. Y′(ω) is real part and Y″(ω) is imaginary part of total admittance, Y(ω). ω is angular frequency and equivalent to 2πf (Kanungo et al.2013b; Manikoth et al.2012). Where, ϵ0 is permittivity of free space (8.85 × 10−12 F/m) and relative permittivity is a unit less quantity. Capacitance of vacuum is denoted as C0 and i is an imaginary unit, (−1)1/2.
Reflection of porosity and dielectric properties on in vitro drug delivery
The influence of dielectric properties and porosity on the drug delivery from hydrogel scaffolds was investigated by comparing with in vitro drug release profile. The percentage of drug released during washing was 40, 30, 34, 28, 27, 23 and 21% for GT, CT and composites in the order from GC0.3 to GC3, respectively. The less interactive drug molecules with polymeric components and drugs residing into micro sized pores are expected to come out during washing. As incorporation of chitosan and genipin cross linkage helps lowering pore size for the composites, they are retaining much amount of drugs into scaffold even after washing. The composite GC0.3, as expected, expels out higher amount of drugs through the scaffold than CT.
This study reveals that cross linking using genipin and increasing the concentration of chitosan leads to the reduction in pore size and increased orientation polarization of gelatin-chitosan polymeric components in hydrogels. Porosity has been explored through the presence of various types of water molecules in hydrogels using thermoporometry. Alternative current impedance analysis gave insights into dielectric properties and yielded information in terms of admittance and relaxation time. We have highlighted how different trends such as slow, immediate and sustained release of drugs from various hydrogels (in vitro drug delivery) correspond to porosity and dielectric properties studied. The results indicate that investigating porosity and dielectric properties would help to design hydrogels for intended drug delivery.
Materials: Gelatin, Type B (bloom strength 125, pI 5.4) for Bacteriology, was purchased from Himedia. Genipin, chitosan, low molecular weight (75% deacetylated) and dialysis tubing cellulose membrane (1.3 inch, M.W cut-off 14,000) were obtained from Sigma Aldrich. Ethanol absolute (99.9%) and acetic acid used throughout the study were of analytical grade.
Preparation of hydrogel composites and drug solution
Gelatin stock solution (3%) was prepared dissolving in warmth double distilled water at 40°C. Dissolution of chitosan was achieved in 2% acetic acid to have the same percentage of gelatin as stock solution. Genipin stock solution (0.1%) was made by dissolving in 40% ethanol. Native hydrogels, gelatin (GT) and chitosan (CT) were prepared separately without addition of cross linking agent to have final concentration 0.5% in water. Various gelatin-chitosan (GC) composites were prepared in such a way to have final concentration of gelatin as 0.5% with different percentage of chitosan. The ratio between gelatin and chitosan in composites was 1:0.3, 1:0.5, 1:1, 1:2 and 1:3 and were labelled as GC0.3, GC0.5, GC1, GC2 and GC3, respectively. Final concentration of genipin was maintained to be 0.01% in all the composites. During composite preparation, the polymer and co-polymer were stirred to get homogenized solution at first and genipin was added drop wise for one hour at 50°C, under constant stirring. The composites thus prepared were kept on stirring for 12 hours at 25°C for the completion of cross linkage. Dialysis was done against double distilled water to purify hydrogels. Catechin was dissolved in 20% ethanol in water to get the final concentration of 12 mM in solution. The tubes containing drug solution were covered instantly with dark brown sheet to be intact of light as much as possible.
Prior to thermoporometry measurement, the intensity of color formed in hydrogels was measured to know degree of cross linkage between polymers with the help of Shimadzu UV- Visible spectrophotometer UV-1800. The experiment was run in the range between 200 and 800 nm at 25°C. Thermoporometry measurement was carried out using Differential Scanning Calorimetry (DSC) by means of Q200 TA instrument. Initially, hermetically encapsulated aluminum pan was utilized for placing the samples and frozen to −25°C. Following this, endothermic thermogram was conducted with the temperature range from −25 to 5°C at a heating rate of 0.5°C/ min. The melting temperature (Tm), onset temperature (Tos) and total enthalpy (Hm) for the phase transition of water in hydrogels were computed using the system generated software. All the experiments were done in triplicate and the mean was taken for calculation. From the thermograms, the pore radii were calculated as given in Results and discussion section below. Scanning Electron Microscopy (Hitachi S-3400 SEM microscope) was used for observation of micro sized pores. All the hydrogels were freeze dried and lyophilized previously to obtain hydrogels in dried form. The surface of the lyophilized samples was captured with the operating condition 15 kV and 300 X magnifications.
AC impedance analysis
AC impedance analysis was carried out to evaluate dielectric properties of native and composites by means of CH instrumental electrochemical analyzer CHI-model 660D (U.S.A). The electrode set up consisted of glassy carbon electrode as working electrode, platinum electrode as counter electrode and saturated calomel electrode as the reference electrode. Open circuit potential (O.C.P) was measured for each sample, prior to AC impedance analysis and it was set as initial electric field (E) during the process. The other operating conditions were temperature (t) = 25°C, low frequency (Hz) = 10−2, high frequency (Hz) = 105, amplitude (V) = 0.005, quite time (s) =2 and (0.1-1 Hz) = 1 Cycles; (0.01-0.1Hz) = 1 Cycles; (0.001-0.01 Hz) = 1 Cycles. All the experiments were done in triplicates and average values were reported.
In vitro drug delivery
Drug solutions were added with all the hydrogels and incubated for 10 hours, at 60 rpm placing in an incubation shaker in dark room. Dialysis membrane was cut into equal length enough to hold the hydrogels. Pretreatment such as submerging the membrane in warmth EDTA solution at 80°C followed by rinsing with hot water was done for ensuring the membrane contains no trace amount of metals and glycerol to protect catechin during in vitro release. Furthermore, it creates well porous network in membrane for diffusion of drug molecules with ease. Washing the hydrogels was done against deionized water to remove the unbound drug molecules at 60 rpm at 25°C for one hour. Subsequently de-loading was also carried out against phosphate buffer saline (1X) raising the temperature to 37°C with 115 rpm. All the arrangements were made to mimic the physiological condition of human body. The amount of drug released (%) during washing and de-loading was calculated from the UV absorbance value around 279 nm.
The authors thank Department of Science and Technology, Govt. of India Fast Track Project for the financial support.
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