Controlled Release of DSBP from Genipin-Crosslinked Gelatin Thin Films
Controlled Release of DSBP from Genipin-Crosslinked Gelatin Thin FilmsAlireza AbbasiDepartment of Chemical Engineering, Ryerson University, TorontoDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
Morteza EslamianDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
Darrick HeydDepartment of Chemistry and Biology, Ryerson University, Toronto, Onatrio, Canada
Dérick RousseauSchool of Nutrition, Ryerson University, Toronto, Ontario, Canada
Controlled release of a marker drug, 4,4′-bis(2-sulfostyryl)biphenyl (DSBP) from genipin crosslinked gelatin thin films,with application to drug delivery by transdermal patches is stud-ied in this paper. A simple method for fabrication of nano-thinfilms, using basic lab equipment is introduced. This method con-sists of two steps: dipping of the substrate in a deposition solu-tion, followed by centrifugation of the substrate. Also, swellingand drug release from thin films is modeled, using the Fick’s sec-ond law of diffusion. The effect of genipin concentration onrelease of DSBP molecules from thin films is investigated,experimentally and numerically. The results show that controlledrelease of DSBP from the genipin-crosslinked gelatin thin filmsis achieved, using various concentrations of genipin in gelatin.
Drug release from gelatin matrices has been the focus ofseveral studies, as gelatin is known to be nontoxic, noncarci-nogenic, biodegradable and biocompatible.[1] Stability of
gelatin hydrogels has been achieved, using several crosslink-ing agents, such as transglutaminase, gluteraldehyde, formal-dehyde and genipin,[2–4] among which gluteraldehyde andformaldehyde are very toxic to the body. Transglutaminase isan enzyme and has applications in sustained drug delivery,medical and dental adhesives, and food industry.[5,6] On theother hand, rather few research papers are available on geni-pin crosslinked gelatin matrices, the subject of this study. In astudy, genipin was employed to crosslink gelatin micro-spheres as a biodegradable drug-delivery system. The resultsconfirmed the ability of genipin as a crosslinking agent.[3]
As a route of drug delivery to the bloodstream, trans-dermal patches, made of thin films, are used to deliver atime released dose of medication, through the skin. Thisdrug delivery route exhibits precise drug release capabili-ties.[7,8] Addition of genipin to any gelatin hydrogel sys-tem, including thin films, has two advantages: It improvesthe stability of the matrix, and also allows the timedrelease of a particular drug. It is known that the swelling ofthe gel and the drug release is affected inversely by theconcentration of genipin.[8]
Evaporation and the spin-coating methods are the twocommon techniques of thin film fabrication. The former iswidely used in fabrication of optical systems, such as anti-reflection coatings on lenses.[9,10] In practice, film deposi-tion of high-boiling polymers may be difficult unless thesubstrate is first heated, as substrate temperature during thedeposition is the key parameter in determining the film struc-ture.[11] The most common method of thin film preparation isthe spin-coating method. Compared to the evaporation
Received 10 April 2008, Accepted 24 June 2008.Address correspondence to Alireza Abbasi, Dept. of Chemical
Engineering and Applied Chemistry, University of Toronto,Toronto, Ontario, Canada; E-mail: [email protected]
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method, it requires less equipment and is generally lessexpensive.[11] The key advantage of the technique is in thecontrol of deposited film structure. However, this methoddoes lead to non-uniformity near the film edges,[12] thisbeing caused by the centrifugal force when spinning.Therefore, one objective of this project was to develop asimple method to prepare gelatin-based thin films that maybe used as matrices for the controlled release of bioactivecompounds, using simple and inexpensive lab equipment.The developed method, called the “dip-centrifuge” method,permits control of the deposited film thickness and edgeuniformity, though thickness may vary along the length ofthe substrate, due to gravity and centrifugal forces.
The 4,4′-bis(2-sulfostyryl)biphenyl (DSBP) is a fluo-rescent whitening agent, widely used by the textile anddetergent industries to whiten fabrics.[13] DSBP was usedin this study as a marker drug, since its intensity can bereadily measured due to its strong fluorescence property,and then correlated with the mass of DSBP moleculeswithin the solution, using a quantifying method called“Fluorescence Spectrophotometry”.[14]
Satisfactory development of controlled release systemsdepends on application of predictive models. Mathematicalmodeling of diffusion of molecules in hydrogels is oftenbased on the free volume theory. Free volume is assumedto be the major factor controlling the diffusion rate of mol-ecules. The diffusion model is based on the probability offinding a hole of a specific volume or larger adjacent tothe diffusant molecule.[15] To the authors’ knowledge,there is no published work on modeling of drug releasefrom crosslinked hydrogel swelling thin films. In one ofthe very few studies, the characteristics of genipincrosslinked gelatin bulk system, such as its swelling anddrug release was investigated.[16]
In summary, the release of entrapped DSBP from thegenipin-crosslinked gelatin thin films, and also the ingressof water into genipin-crosslinked gelatine disks and filmsare simulated by Fick’s second law of diffusion. Also, theeffect of genipin concentration variation on drug releasecharacteristics of gelatin films is studied, experimentallyand numerically. Atomic Force Microscopy (AFM) isused to visualize the film surface microstructure andtopography, and to calculate the surface roughness.
MATERIALS AND METHODS
Preparation of Stock Solutions
500 mL gelatin (Type A, Bloom 300, Sigma-AldrichCo., Oakville, ON, Canada) stock solution was preparedby dispersing gelatin powder in distilled de-ionizedwater (DDW) at 50°C with continuous stirring for
30 min, pH adjusted to 5 (0.5 M HCl). 200 mM genipin(Challenge Bioproducts Co., Taichung, Taiwan) stocksolution was prepared by dissolving genipin powder in60% (v/v) ethanol, as described by Yao et al.[17]. 500 mLDSBP (Ciba Co., Mississauga, ON, Canada) stock solu-tion with concentration of 1.75E-7 mol/lit, was preparedby dilution of DSBP liquid in acetate buffer, pH adjustedto 5 (0.5 M HCl).
Preparation of DSBP-Loaded Genipin-Crosslinked Gelatin Thin Films
Figure 1 outlines the preparation method of the thinfilms. Gelatin/genipin solutions were prepared by mixing20 mL of 10 wt % gelatin solution at 60°C with 0.125,0.25, 0.5, 0.75, 1 and 2 wt % of genipin at 25°C, while stir-ring the solution very slowly in order to avoid the foamformation. The gelatin/genipin solution was then pouredinto 2 × 2 × 8 cm slide containers (Fisher Scientific,Ottawa, ON, Canada). Clean 25 × 76 mm slides (FisherScientific, Ottawa, ON, Canada) were immersed and tiltedinside the containers for 1 min to let the solution getabsorbed in the glass surface. The slides were taken outand placed upside down in centrifuge tubes to be centri-fuged at 1600 RPM for 2.5 min at room temperature. Theorientation of slides in centrifuge tubes was kept constantto avoid unequal centrifugal force acting along the widthof the slides. After 24 h, 13 mL of DSBP, at concentrationof 1.75E-7 mol/lit of stock solution was mixed with thegelatin/genipin solutions, made with different concentra-tions of genipin. The gel times of such systems have beenshown to be on the order of several hours.[18] Then the gelslides were immersed and tilted inside the slide containersagain and held for 50 min (This stage is not shown inFigure 1). This time is enough for the DSBP to diffuse tothe gel, and ensure that the entire system has reached equi-librium. Subsequently the slides were rinsed with distilledwater and placed at room temperature for 30 min, to let thewater completely evaporate from the surface of the slides.All gel slides were prepared in triplicate.
Preparation of Genipin-Crosslinked Gelatin Disks
Genipin-crosslinked gelatin disks, which are muchlarge in size than the thin films, were prepared to accuratelymeasure the swelling behaviour of genipin-crosslinked gel-atin and relate and compare it to that of the films. To pre-pare the disks, the gelatin/genipin stock solutions werepoured into plastic screw-capped tubes (moulds), pre-lubri-cated with a thin layer of cooking spray, and then allowedto set for 24 h at room temperature. Once set, gels were
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prepared in disk forms (3 cm in diameter and 2 mm inheight). All gel disks were prepared in triplicate.
Film Morphology
The morphology of the genipin-crosslinked gelatinfilms as well as their thickness and roughness at differ-ent concentrations of genipin were examined using anAFM (Bioscope, Digital Instruments, Veeco Metrology,CA, USA), using the tapping mode at surface scan of2500 × 2500 × 50 nm.
Visually, the films were only slightly visible to thenaked eye. The height of the freshly-made genipin-crosslinked gelatin films was measured by the AFM.Detail of the measurement process is illustrated inFigure 2. Each slide was divided in three zones along itslength. Using a stainless steel razor blade, the gels were
Figure 1. ‘Dip-centrifuge’ preparation method of genipin-crosslinked gelatin films. Note that for the best result, the bottom edgeimmersed in the solution should be the same as the edge, mounted closer to the rotating axis of the centrifuge machine.
Figure 2. Thickness measurement of genipin-crosslinkedgelatin films.
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scored at distances of 0.2, 1.0 and 1.8 cm from the baseof the slide. In each zone, the thickness was measuredacross the film at three locations. All gels slides tested intriplicate.
Swelling Index
Genipin-crosslinked gelatin disk samples were ini-tially weighed (W1), then immersed in 15 mL of acetatebuffer, pH 5, (NaCl) (0.5 M) at ambient temperature forthe same duration as the release experiments. Sampleswere blotted dry and weighed (W2), after each time inter-val. The swelling index (SI), was calculated using the fol-lowing Equation:[17]
All measurements were made in triplicate.
DSBP Release Experiments
DSBP release experiments were carried out in tripli-cate in a beaker containing 40 mL of buffer solution (ace-tate 0.01 M, pH 5, 0.5 M NaCl) at room temperature. Thegel slides loaded with DSBP were immersed in the buffersolution, while stirring continuously in order to avoidboundary layer formation over the slide surface. Duringthe release experiments, samples of the buffer solutionwere drawn from the beaker at certain times, and the fluo-rescence intensity generated by the released DSBP wasmeasured, using a luminescence spectrometer (Model LS50B, Perkin-Elmer Life and Analytical Sciences, Inc.,Waltham, MA, USA). The measured sample was pouredback into the buffer solution.
MATHEMATICAL MODEL
DSBP release from the genipin-crosslinked gelatinfilms and the ingress of water into genipin-crosslinked gel-atin disks and films were simulated by Fick’s second lawof diffusion, based on the free volume theory. The DSBPloaded genipin-crosslinked gelatin matrix was film layerin shape, swelled uniformly in the axial direction, and wasassumed to be ideal. Release and swelling is considered inone direction (direction normal to the film thickness).Thus, the matrix swelled during water uptake, and thedimension of the matrix changed from the initial thickness(z0) to the new thickness (zt) (Figure 3). Fick’s second law
in Cartesian coordinate system in one dimension form is asfollows:[19]
A Fujita-type model for the exponential dependenceof the diffusivities of water and DSBP (Di) was used:[20]
In Equation (3),Di,eq is the diffusion coefficient of water orDSBP in the fully swollen gelatin matrix, bi is a dimen-sionless constant that characterizes the concentrationdependence of water or DSBP diffusivity,C1,eq is the equi-librium water concentration in fully swollen gelatinmatrix, and C1 is the water concentration (Equation 2 fori = 1). The values for D1,eq D2,eq, b1, and b2 were deter-mined by fitting the numerical solutions of Equation (2) toexperimental data.
Initial and Boundary Conditions
The initial and boundary conditions for DSBP-loadedgenipin-crosslinked gelatin thin films and genipin-crosslinked gelatin disks are similar. For the thin films,initially, the matrix is dry, and the DSBP is uniformly dis-tributed throughout the matrix. The first boundary condi-tion is derived assuming that the surface of the film is aperfect sink for the DSBP, i.e. the concentration of DSBPon the film surface is zero, and the concentration of water
SIW W
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Figure 3. Schematics showing the swelling of genipin-crosslinked gelatin for (a) films, and (b) disks, used formathematical modeling.
(a)
(b)
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Controlled Release of DSBP from Genipin-Crosslinked Gelatin Thin Films 553
on the film surface is equal to the equilibrium concentra-tion of water during the entire release process. To derivethe second boundary condition, the diffusion fluxes ofwater and DSBP in the interface of the slide and the thinfilm are considered zero.
The initial and boundary conditions for genipin-crosslinked gelatin disks are as follows: Initially the diskis dry, i.e. the concentration of water is zero everywherewithin and on the disk. The first boundary condition isobtained from the fact that the water concentration on thedisk surface is equal to the equilibrium concentration dur-ing the entire water uptake process. The second boundarycondition is derived from the symmetry of the disk, whichresults to zero diffusion flux of water in the middle of thedisk. Initial and boundary conditions of similar systemsare mathematically shown in [16].
RESULTS AND DISCUSSION
Film Thickness
The results revealed that along the 2 cm length of thefilm, the film thickness linearly increased from 350 to 630nm. During the centrifuge process, the slides came to ahorizontal position, with the side under consideration fac-ing downward. The reason that the film thicknessincreases with an increase in the radius of rotation (dis-tance along the film measured from the axis of rotation ofthe apparatus) is that the centrifugal force acting on thefilm increases with increase of the radius of rotation.
It was observed that genipin concentration had no effecton the local film thickness. This is because the centrifugal
force acting on the film, and also the gravity force areresponsible for shaping the film. The crosslinking forceappears after several hours from the formation of the matrix.
Film Morphology and Roughness
Figure 4 shows microstructure of genipin-crosslinkedgelatin thin films, at surface scan of 2500 × 2500 × 50 nmfor two concentrations of genipin. The presence of genipinaltered the surface morphology. Visually, Figure 4 showsthat an increase in the concentration of genipin, leads tothe formation of a smoother film surface. To investigatethis effect quantitatively, the surface roughness (RMS) ofthe genipin-crosslinked gelatin films were calculated;Figure 5 shows the roughness profile (RMS) as a functionof genipin concentration. Each data point is the average ofthree measurements, and the error bars show the standarddeviation of the data points. It is observed that the surfaceroughness decreases with an increase in the concentrationof genipin. For genipin concentrations of greater than 1%(w/w), the graph showing the variation of roughness withgenipin concentration has a plateau. An increase in theconcentration of genipin results in a better crosslinking;therefore, the number of free molecules in the matrixdecreases. This might be the reason for having a smoothersurface with increase of genipin concentration.
Water Uptake in Genipin-Crosslinked Gelatin Disks
Figure 6 shows the experimental and numerical dataof variation of relative water uptake with time, in gelatin
Figure 4. Surface microstructures of genipin-crosslinked gelatin films, taken by AFM, for genipin concentrations of 0.25 and 2.00wt %. The scale is 2500 × 2500 × 50 nm. Note that with increase of genipin concentration, a smoother surface is obtained.
0.25 wt% genipin 2.00 wt% genipin
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disks for different concentrations of genipin. For eachgenipin concentration, the constants of the Fick’s law,D1,eq and, b1,(Equation 2) were chosen such that thenumerical results best fits the experimental data. The dif-fusion coefficient of water in gel disks, D1,eq and b1 fordifferent genipin concentrations, are shown in Table 1.The coefficient of determination, R2, for all systems isgreater than 0.99. Table 1 shows that with increase ofgenipin concentration, the diffusion coefficient of water atequilibrium decreases. Figure 6 shows the numerical solu-tions in good agreement with the experimental data. Notethat for the lowest concentration of genipin (0.25 wt %),after 1200 min, the water concentration in the disk reachesthe equilibrium concentration. With increase of genipinconcentration this time decreases significantly so that for a
genipin concentration of 2 wt %, the equilibrium concen-tration is reached after a few minutes.
When the genipin-crosslinked gelatin is immersed intowater, it absorbs water, and consequently swells. Stretchingof pores within the gel, results in a wider pore size distribu-tion. The water concentration inside the genipin-crosslinkedgelatin matrix is dependent on the diffusivity of water,resulting in a concentration-dependent diffusivity. It is obvi-ous that with increase of the amount of genipin concentra-tion in gelatin, the available free space in the systemdecreases. As a result, water uptake decreases, and the sys-tem comes to equilibrium faster (see Figure 6).
Swelling Kinetics in Genipin-Crosslinked Gelatin Disks
To investigate the swelling kinetics, the time variationof swelling of genipin-crosslinked gelatin disks for differentconcentrations of genipin was calculated, numerically. Itwas assumed that no volume contraction occurred duringswelling and water ingress was considered to be axially, i.e.swelling was along the z axis only (see Figure 2). The over-all shape of the swelling curves (not shown here) was rathersimilar to those of water uptake curves, shown in Figure 6.The results showed that for systems with higher concentra-tion of genipin, less swelling occurs and the matrix comes toequilibrium faster. This is because according to the free vol-ume theory, more water ingresses inside, along with moreswelling in gel matrix for less genipin concentration.
Water Uptake in Genipin-Crosslinked Gelatin Films
D1,eq and β1 values calculated for genipin-crosslinkedgelatin disks were used to predict the swelling time andswelling kinetics of genipin-crosslinked gelatin films. Theinitial thickness of the gel films used for the modeling was475 nm. Table 2 shows the swelling time and swelled thick-ness of the films. With increase of genipin concentration,
Figure 5. Surface roughness (RMS) profile of genipin-crosslinked gelatin films as a function of genipin concentration.Each data point is the average of three measurements and theerror bars represent the standard deviation of the data.
Figure 6. Water uptake profiles of the genipin-crosslinked gelatindisk matrices, obtained by the Fick’s second law of diffusion(continuous lines). The experimental data points are also shown.
Table 1 D1,eq and b1 values for genipin-crosslinked gelatin gels
Controlled Release of DSBP from Genipin-Crosslinked Gelatin Thin Films 555
the swelling time and swelled thickness decrease, consis-tent with the results obtained for gelatin disks. The resultsshow that the time for film swelling is much shorter thanthat for releasing of DSBP molecules,[21] confirming thatthe genipin-crosslinked gelatin films swell completelybefore the release of DSBP through gel films starts.
DSBP Absorption in Genipin-Crosslinked Gelatin Films
In order to find the amount of absorbed DSBP in eachgelatin film, the films were immersed in 8 M urea solu-tion. Urea breaks hydrogen bonds, and substantially weak-ens hydrophobic interactions. The details of the procedureare explained elsewhere.[21,22] Table 3 lists the totalamount of absorbed DSBP to gel films for different con-centrations of genipin. The total amount of absorbedDSBP is dependent on the genipin concentration in thegel, and with increase of genipin concentration itincreases. These results may be interpreted as there beingsubstantial electrostatic interaction between DSBP mole-cules and gelatin matrix. DSBP is a negatively-chargedmolecule with a sulfate group at each end. If a positively-charged molecule is present in the vicinity, DSBP will beelectrostatically attracted toward it. Gel formation in gela-
tin is the result of helix formation of parts of the macro-molecules, where hydrogen bonding plays an importantrole. Gelation of gelatin solutions is primarily determinedby the amount of the amino acids proline (PRO) andhydroxyproline (HYP), whereas also the amino acid gly-cine (GLY) plays an important role.[23] Genipin crosslinksbreak open the alpha helices inside gelatin exposing addi-tional positively charged amino acid R-groups.[24,25] In aneutral to acidic environment, these amino acids are posi-tively charged, leading to binding of negatively-chargedDSBP molecules to gelatin, due to electrostatic interac-tions. The exposure of additional genipin in the solutionand consequently amino acids leads to an increase in theelectrostatic attraction of DSBP molecules towards gelatinmatrix.
DSBP Release Through Genipin-Crosslinked Gelatin Films
Figure 7 shows the time variation of DSBP release forvarious genipin concentrations. Using the same curve-fit-ting procedure as that of Figure 6, the numerical solutionsof the Fick’s second law, equation (2), for each genipinconcentration were fitted to the experimental data. Figure 7shows that by varying the concentration of genipin, con-trolled release of DSBP from the gelatin matrix isachieved. Also note that, with increase of genipin concen-tration, the release of DSBP is delayed. For a film withgenipin concentration of 1% (w/w), it takes about 1 h forDSBP molecules to be depleted from the gelatin film.
The DSBP diffusion coefficients calculated by curve-fitting procedure are shown in Table 4. Table 4 shows thatwith increase of the genipin concentration, the diffusioncoefficient of DSBP decreases. Diffusion coefficient of
Table 2 Swelling time and swelled thickness of genipin-crosslinked
Figure 7. Time variation of DSBP release from genipin-crosslinked gelatin films at various genipin concentrations. Thecontinuous lines are the model results and the single points arethe experimental data.
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DSBP is independent of water concentration in the matrix;however the transfer kinetics of diffusing DSBP moleculesdepends greatly on the molecular size, shape and pore size,with small pore sizes leading to lower diffusion coeffi-cients. Pores that are larger than the diffusing moleculewill permit diffusion with little or no resistance, whereasdiffusing species larger than the pores will encounter resis-tance against their flow as they entangle with the matrixmesh. The jump from one pore in the biopolymer structureto another for a given pore size distribution will be easierfor smaller than for larger molecules.[26] Hence, anincrease in genipin concentration results in smaller poresize, and, therefore smaller diffusion coefficients of DSBP(Table 4) and water (Table 1).
CONCLUSIONS
A simple method for fabrication of thin films, usingcommonly-available laboratory equipment was developedand employed to prepare genipin-crosslinked gelatin thinfilms. The release of a very effective marker drug, DSBP,from the thin films was investigated experimentally andnumerically. Fick’s second law of diffusion was used tosimulate the water and DSBP diffusion within the gelatinthin films and disks. The numerical solutions were fittedwith the experimental data to obtain the diffusion coeffi-cients of water and DSBP. Using these coefficients, thepredicted marker release profiles were in a very goodagreement with the experimental data at different gel com-positions. For practical applications, this one-dimensionalmodel is a simple tool to examine the timed release of aparticular drug from a crosslinked gelatin thin film.
The release mechanism of DSBP from genipin-crosslinked gelatin matrix was elucidated: The release ofDSBP molecules depends on the free available volume.Primarily, water concentration gradient between the geland environment makes water ingress into the gel. As aresult, the biopolymer chains expand, leading to swelling.The swelling alters the dimensions of the pores. Ingress of
water stops when the chemical potential of water withinthe hydrogel becomes equal to that of the surroundings. Itwas found that swelling occurs so fast that diffusivity ofDSBP is independent of water concentration in the gelmatrix. It was also observed that by varying the concentra-tion of genipin, controlled release of DSBP from the gela-tin matrix would be achieved. With increase of genipinconcentration, the surface roughness of the film decreased;also, the release of DSBP was delayed.
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Table 4 DSBP diffusion coefficients in fully-swelled genipin-
crosslinked gelatin films
Genipin concentration(wt %)
DSBP diffusion coefficient D2,eq × 1012 (cm2/s)
0.25 7.700.50 3.050.75 1.851.00 1.302.00 1.10
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