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Silk fibroin aerogels for drug delivery applications
Accepted Manuscript Title: Silk fibroin aerogels for drug delivery applications Author: Michael A. Marin Rajendar R. Mallepally Mark A. Mc Hugh PII: DOI: Reference:
S0896-8446(14)00109-0 http://dx.doi.org/doi:10.1016/j.supflu.2014.04.014 SUPFLU 2962
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
26-2-2014 23-4-2014 24-4-2014
Please cite this article as: M.A. Marin, R.R. Mallepally, M.A. M, Silk fibroin aerogels for drug delivery applications, The Journal of Supercritical Fluids (2014), http://dx.doi.org/10.1016/j.supflu.2014.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Applications
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Silk Fibroin Aerogels for Drug Delivery
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Michael A. Marin1, Rajendar R. Mallepally,*1, and Mark A. McHugh1,2 Department of Chemical and Life Science Engineering, Virginia Commonwealth University,
Materials Sciences Division, VCU Reanimation Engineering Science Center, Virginia
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Richmond, VA 23284.
Commonwealth University Medical Center, Richmond, VA 23298.
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ABSTRACT: Silk fibroin (SF) is a natural protein, derived from the Bombyx mori silkworm. Silk fibroin based porous materials are being extensively investigated for biomedical applications,
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due to their biocompatibility and biodegradability. The results presented here demonstrate the potential of SF aerogels as drug delivery devices for the extended release of ibuprofen, a candidate drug. SF aerogels are loaded with of ibuprofen using supercritical carbon dioxide (scCO2) at 40 °C and 100 bar. Differential scanning calorimetry of the ibuprofen-loaded SF aerogels indicates that the ibuprofen is amorphous. Scanning electron microscopy and nitrogen adsorption/desorption analysis are used to investigate the morphology and textural properties. Phosphate buffer solution (PBS) soaking studies, at 37 °C and pH 7.4, reveal that SF aerogels do not swell nor exhibit any weight loss for up to six hours, the lifetime of the release measurements performed in the present study. In vitro ibuprofen release in PBS, at 37 °C and pH 7.4, occurs over a six-hour period when the ibuprofen is loaded in SF aerogel discs that are 1.4 cm in
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diameter and 0.85 cm in height. In contrast, the dissolution of the same amount of pure ibuprofen occurs in 15 minutes. Furthermore, the release of ibuprofen from these SF aerogel discs are modeled using the Ritger-Peppas model which indicates that ibuprofen release follows Fickian
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diffusion.
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KEYWORDS: Silk fibroin, Drug loading, Drug delivery, Protein hydrogel, Protein aerogel,
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Supercritical impregnation 1. Introduction
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Hydrogels are three-dimensional hydrophilic polymer networks that are being extensively investigated for biomedical applications such as tissue regeneration matrices [1, 2], cell scaffolds
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[3, 4], and drug delivery devices [5, 6]. These gels exhibit unique properties such as an open-
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pore network, structure tunability, and biocompatibility [6]. However, most hydrogels lack
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mechanical strength, which can be a problem during handling [7]. This low mechanical strength and subsequent handling issue can often be overcome by air drying the hydrogel to create a
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xerogel, freeze drying the hydrogel to create a cryogel, or drying the aerogel with supercritical carbon dioxide (scCO2) to create an aerogel. Air drying is the least complex method, but with this method the open-pore network of the hydrogel structure collapses due to capillary forces from evaporating water [8]. In contrast, freeze drying preserves the open-pore, hydrogel network by eliminating capillary forces as water is removed from the matrix by sublimation. However, the formation of ice crystals can destroy fine pore structure [9]. The drawbacks associated with both air drying and freeze drying are eliminated by using scCO2 drying, thus preserving the initial hydrogel structure.
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Once the aerogel is synthesized, several methods are available for loading a drug into the aerogel, including soaking the aerogel in a liquid solution or contacting the aerogel with a gas or supercritical fluid saturated with the drug [10, 11]. Loading the aerogel via soaking makes it
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difficult to fill small diameter pores with a liquid phase due to surface tension limitations and once loaded, an additional drying step is required. As an alternative, loading the aerogel via a
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saturated gas eliminates both the surface tension limitations and the drying step, but this type of
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processing can still be problematic due to the low drug solubility in a gas phase. A supercritical fluid (SCF) solvent can overcome many of the limitations associated with liquid and gas phase
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loading methods since an SCF solvent has a very low viscosity and a low solid-SCF surface tension, which means that SCF solvent, saturated with drug, can easily penetrate small pores. In
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that observed with a gaseous solvent [12].
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addition, an SCF solvent can have a drug solubility level several orders of magnitude higher than
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A fair amount of research has been reported on the loading of silica aerogels using supercritical fluid solvents [13-15]. However, these siliceous materials lack biodegradability,
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which is a primary prerequisite for drug delivery applications. Researchers have overcome this disadvantage by creating aerogel drug delivery devices consisting of polysaccharides [16-24] or covalently-linked proteins [9]. In this work, silk fibroin (SF) protein, from the cocoon of a Bombyx mori silkworm, is used to synthesize an aerogel for use as a drug delivery carrier. Although researchers have previously synthesized certain types of SF drug delivery matrices [18], to the best of our knowledge, there are no prior reports specifically on the synthesis and use of SF aerogels as a drug delivery devices. In the present study scCO2 is used to load ibuprofen, a candidate drug compound, into the SF aerogels. Scanning electron microscopy and N2 adsorption/desorption are used to determine
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the micro and macro structure, surface area, pore size, and pore volume of the SF aerogels. Phosphate buffer solution (PBS) soaking studies are performed to determine the amount of aerogel volume change and weight loss. The release rate of ibuprofen from the SF aerogels is
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measured and contrasted with the dissolution rate of pure ibuprofen to ascertain the impact of
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aerogel morphology on the delivery of ibuprofen.
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2. Materials and Methods
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2.1 Materials
Bombyx mori silkworm cocoons are purchased from Aurora silk (Portland, Oregon,
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USA). Sodium carbonate, ibuprofen, and lithium bromide are purchased from Sigma Aldrich,
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USA. All solvents are analytical grade and used without further purification.
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2.2 Preparation of silk fibroin solution and aerogels Cocoons are treated with an aqueous solution of 0.02 M Na2CO3 (1% w/v) at 90 °C for 60 minutes. Degummed fibers are recovered and washed with boiling water to remove the sericin protein that coats the SF protein. These fibers are stretched and dried overnight at 40 °C and are dissolved in 9.3 M aqueous LiBr solution (10% w/v) at 65 °C for four hours. The fiber-LiBr aqueous solution is cooled to 25 °C and dialyzed (Snakeskin Dialysis Tubing, Thermo Scientific, USA, MWCO 3,500 Da) against distilled water for three days (2.5% v/v), with every day water replacement. An aqueous 4 wt% SF solution is poured into a 10 mL glass beaker that is placed in a 300 mL stainless steel vessel (Parr Instruments, USA), which is heated to 40 °C and pressurized
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overnight with CO2 at 100 bar. The stainless steel vessel is slowly depressurized over 1.5 hour period and the recovered SF hydrogel is converted into an alcogel by soaking in aqueous solutions of increasing alcohol concentration (20, 40, 60, and 80 wt%) for 30 minutes each,
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followed by soaking in pure ethanol overnight. Ethanol is removed from the SF alcogel using scCO2 at 40 °C and 100 bar to create the SF aerogel. The volume of the SF aerogel is ~10-to-
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20% smaller than the SF hydrogel. It is worth noting that no attempt is made to optimize the
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processing of the SF aerogel, which should be investigated in the future in a similar way as done
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by Della et al. [25].
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2.3 Ibuprofen loading in silk fibroin aerogels by supercritical CO2
Ibuprofen is loaded into the aerogel using scCO2 following the method reported by Shen
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et al. 2008[26]. SF aerogel is first dehydrated at 100 °C under vacuum for two hours at -762 mm
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Hg and then placed into an 80 mesh stainless steel basket that is attached to an impeller rod
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located in a stainless steel vessel. Excess solid ibuprofen is placed in the bottom of the vessel so that scCO2 remains saturated during this loading process, which is run for two hours at 100 bar at 40 °C with stirring. Ibuprofen is expected to partition between the CO2-rich phase and the SFrich phase [26, 27]. The loading of ibuprofen, 20.6 ± 4.1 wt% (mean ± standard deviation, n = 4), is determined gravimetrically and is cross checked using TGA as subsequently described.
2.4 Silk fibroin aerogel characterization Aerogel morphology is determined using scanning electron microscopy (SEM) (HITACHI SU-70). The aerogels are lightly dabbed on double sided carbon tape stuck to
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aluminum stubs, then coated with ~10 nm of platinum via spin coating (Denton Vacuum, USA, Model: Desk V TSC). Aerogel surface area, pore size, and pore volume are determined using nitrogen
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adsorption/desorption measurements at 77 K (Quantachrome Instruments, Nova 2200e).
Approximately 50 mg of aerogel is heated at 120 °C under vacuum for two hours to dehydrate
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the aerogel before performing the adsorption/desorption measurements. The specific surface
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area of the aerogel is calculated with the multipoint Brunauer–Emmett–Teller (BET) model in the relative pressure range of 0.05 to 0.30, and the pore size distribution is calculated
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with the Barrett-Joyner-Halenda (BJH) model using a desorption isotherm.
Ibuprofen loading in the aerogel is determined using Thermal Gravimetric Analysis
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(TGA) (Perkin–Elmer USA, Model Pyris 1 TGA). The furnace is continuously flushed with
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nitrogen gas at a flow of 3 L/hour. The aerogels are rapidly heated to 80 °C, held at this
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temperature for 15 minutes, and then rapidly heated to 200 °C and held for 30 minutes. In both cases the heating rate is 100 °C/minute.
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The physical state of the ibuprofen in the aerogel is determined using differential scanning calorimetry (TA instruments, Q10). The aerogel sample is heated at 10 °C/minute from 40 to 135 °C. Sample purge flow is set to 50 mL/minute, and resultant data from the first heat are analyzed using Universal Analysis 2000 software.
2.5 Silk fibroin aerogel soaking studies The volume change of an SF aerogel disk is determined by noting the change in the length and diameter of the disk submerged in 50 mL of well-stirred PBS at 37 °C and pH 7.4. Before immersion in PBS, the aerogel is dried at 100 °C under vacuum for two hours. The SF
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aerogel is removed from the solution for immediate imaging and dimension measurement after one minute and 360 minutes. It is worth noting that macroscopic ibuprofen crystals are readily apparent to the naked eye when the loading is increased above ~60 wt%. When ibuprofen is
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loaded “in” the aerogel the ibuprofen is amorphous due to interactions with the fibroin matrix. Once the internal surfaces are saturated with ibuprofen, the excess ibuprofen precipitates as a
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crystalline solid since there are no more sites available to inhibit crystallization. Several literature
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studies have reported similar observations and interpretations on the solid form of ibuprofen or
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alkyl ketene dimer in matrices [27-29].
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2.6 In vitro drug release study
A known amount of ibuprofen-loaded SF aerogel or pure ibuprofen is put into a stainless
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steel basket made with 80 mesh screen. The basket is fully immersed in 50 mL of well-stirred
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PBS at 37 °C and pH 7.4. Perfect sink conditions are maintained during the release experiments
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since the 50 ml of PBS remain below 10% of the 6.02 mg/mL maximum solubility of ibuprofen [30]. Aliquots of PBS are removed at specific time intervals to quantify the release rate of ibuprofen and the same amount of fresh PBS at 37 °C is added back to the parent solution to maintain perfect sink conditions. The ibuprofen solution concentration is quantified using UVVis spectroscopy from the absorbance at 264 nm.
3. Results and Discussion 3.1 Silk fibroin aerogel characterization
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Figure 1 shows the surface area, pore size, and pore volume of the SF aerogels synthesized in this study as determined by adsorption/desorption isotherms. The isotherm exhibits type IV behavior and shows a significant hysteresis indicating the presence of
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mesopores. Figure 2 shows the pore size distribution of a representative SF aerogel which ranges from ~5 to 130 nm. The SF aerogels synthesized in this study have surface areas of 424 ± 75
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m2/g and densities of 0.058 ± 0.001 g/mL (mean ± standard deviation, n = 3). These SF aerogels
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have textural properties very similar to those exhibited by other whey protein aerogels [9].
DSC is used to probe the physical state of ibuprofen in the SF aerogel matrix. Figure 3
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shows DSC curves for pure ibuprofen, pure SF aerogel, and ibuprofen-loaded SF aerogel. The pure ibuprofen curve in Figure 3A has a sharp endothermic peak at ~77 °C, which is from the
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melting of crystalline ibuprofen. The pure SF aerogel curve in Figure 3B does not contain any
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peaks indicating that the unloaded aerogel does not have any crystallinity. However, the curve
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for the SF aerogel loaded with ~19 wt% ibuprofen exhibits a small endothermic peak at ~77 °C, indicating that more than 99.98 wt% of the loaded ibuprofen is amorphous. Evidently the interactions between ibuprofen and the SF aerogel matrix effectively inhibit the crystallization of ibuprofen. The results obtained in this study are consistent with the results reported by Kazarian et al. who loaded up to 30 wt% of amorphous ibuprofen into poly(vinyl pyrrolidone) using scCO2 assisted impregnation [27].
Figure 4 shows the weight loss curves for pure ibuprofen, SF aerogel, and ibuprofenloaded SF aerogel obtained by TGA. The dashed line shows the temperature profile which includes a 15 minute isothermal step at 80 °C to evaporate any water, followed by a 30 minute
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isothermal step at 200 °C to decompose and evaporate ibuprofen. SF aerogels synthesized in this study typically have 4-5 wt% of water that evaporates at 80 °C, and another 1-2 wt% of SF weight loss during the remainder of the TGA run. Figure 4 also shows that pure ibuprofen
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decomposes and evaporates rapidly at 200 °C and that the ibuprofen-loaded SF aerogel loses ~25 wt% during the 200 °C isothermal step. This weight loss is exactly matches the amount of loaded
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ibuprofen determined gravimetrically.
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The micro and nano structures of the SF aerogels are investigated by analyzing SEM images of representative SF aerogels and ibuprofen-loaded SF aerogels. The SEM images in
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Figure 5 (A-D) show the nanofibrous network of the loaded and unloaded aerogels. This fibrous network is stabilized by crystalline beta sheet conformations formed from inter- and
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intramolecular hydrogen bonding and hydrophobic interactions [31]. The crystalline beta sheets
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are formed during the sol-gel transition of silk fibroin as determined from a Fourier transform
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infrared spectroscopy analysis not shown here. There is a strong positive correlation between the pore sizes observed in the SEM images and the pore size distribution data obtained from the N2 adsorption/desorption analysis shown in Figure 2. It is worth noting that macroscopic ibuprofen crystals are readily apparent to the naked eye when the loading is increased above ~60 wt%. When ibuprofen is loaded “in” the aerogel the ibuprofen is amorphous due to interactions with the fibroin matrix. Once the internal surfaces are saturated with ibuprofen, the excess ibuprofen precipitates as a crystalline solid since there are no more sites available to inhibit crystallization. Several literature studies have reported similar observations and interpretations on the solid form of ibuprofen or alkyl ketene dimer in matrices [27-29]. No attempt is made to determine the
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saturation solubility of ibuprofen in the aerogel, above which pure ibuprofen crystals are
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observed.
3.2 Silk fibroin aerogel soaking studies
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Figure 6 shows images and dimensions of the SF aerogels after soaking in PBS at 37 °C ratio of ~1.75, whereas after one minute in
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and pH 7.4. Initially the SF aerogel has a
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solution the ratio reduces ~1.65 where it remains for the next six hours. Although the
ratio remains constant during the PBS soaking studies, the aerogel has already shrunk by ~46 %
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of its initial volume. The aerogel shrinkage is likely due to a combination of effects. Shrinkage can occur due to attractive ionic interactions between de-protonated carboxylic acid groups and
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protonated amino groups. Shrinkage can also occur due to the collapse of macropores by capillary forces exerted from rapid water transport into the matrix. It is likely that both
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phenomenon occur simultaneously resulting in matrix collapse. Further studies are in progress to elucidate the cause behind this aerogel shrinkage. It is worth noting that gravimetric analysis of the SF aerogel after the soaking experiment reveals no aerogel weight loss. Therefore, the release of ibuprofen is likely only based on diffusion from the matrix since dimensional changes in the SF aerogel matrix are not observed.
3.3 In vitro drug release studies In vitro ibuprofen release studies are performed at perfect sink conditions to ensure there are no concentration effects on the diffusion and dissolution of ibuprofen. Figure 7 shows the release profile for pure ibuprofen and ibuprofen-loaded SF aerogels in PBS at 37 °C and pH 7.4.
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Pure ibuprofen is completely dissolved within 15 minutes, whereas ibuprofen from the SF aerogel is released over a period of approximately 360 minutes. Note that the ibuprofen loaded in the SF aerogels exhibits a release profile composed of two different rates. Initially, ~75 wt% of
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the ibuprofen is released in 100 minutes followed by a significantly slower release of ~15 wt% of the ibuprofen from 100 to 360 minutes. The remaining 10 wt% of the ibuprofen is either strongly
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bound to the SF aerogel matrix or is trapped in collapsed pores in the matrix and would not be
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released until the aerogel degraded. The ibuprofen-loaded SF aerogel release profiles are similar
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to those observed for ibuprofen loaded alginate and eurylon starch aerogels [16].
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Ibuprofen release behavior from the silk fibroin aerogels is analyzed using the Ritger-
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Peppas model, which is given by:
Mt kt n M
(1)
where Mt/M∞ is the fractional weight release, k is a constant, t is time, and n is the diffusional exponent that depends on the release mechanism [32]. This model is valid for the first 60 wt% of total drug release and the model assumes no swelling of the drug delivery device, which is an accurate assumption given that the dimensions of the SF aerogel remains constant one minute after immersion in PBS. The diffusional exponent in equation 1 is determined by minimizing the mean average percent deviation (MAPD) between predicted and experimental release of ibuprofen from the SF aerogel. The MAPD is given by:
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1 n Experimental releasei Predicted releasei 100 n i1 Expermental releasei
(2)
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MAPD
where n is equal to the number of data points. A diffusional exponent of 0.43 ± 0.03 (mean ±
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standard deviation, n = 3) and an MAPD of 1.1 ± 0.3 % (mean ± standard deviation, n = 3) is
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obtained when fitting the data to equation 1. This value of the diffusional exponent obtained for the release of ibuprofen from the disk shaped SF aerogels suggests that the diffusion is Fickian
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[32].
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4. CONCLUSIONS
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The high surface area and open pore network of SF aerogels, along with their
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biocompatibility and biodegradability [33], make them attractive drug delivery devices. The present work demonstrates the ability of silk fibroin aerogels to deliver a candidate drug for an
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extended period of time. Ibuprofen loading using supercritical CO2 resulted in SF aerogels loaded with ~21 wt% of ibuprofen. Release of ibuprofen from SF aerogels is found to be governed by Fickian diffusion. Further studies are in progress to demonstrate the effect of tailoring the morphological and textural properties of SF aerogels to attain a drug delivery device that yields different target drug release profiles.
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AUTHOR INFORMATION Corresponding Author
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* E-mail: [email protected]
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge partial support for this work from the Office of Naval Research from Grant N000140710526. We also express thanks to the reviewers for their
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scientific inputs.
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Figure 1 . Typical N2 adsorption (●)/desorption (○) isotherm for the SF aerogels synthesized in this study.
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Figure 2. Typical pore size distribution of SF aerogels synthesized in this study.
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Figure 3. Differential scanning calorimetry curves. (A) pure ibuprofen and (B) pure SF aerogel (dashed line) and ibuprofen-loaded SF aerogel (solid line) obtained in this study. All measurements are performed in an open pan.
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Figure 4. Weight loss curves for pure ibuprofen (△), SF aerogel (○), and ibuprofen-loaded SF aerogel (□). The dashed line represents the TGA temperature profile.
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Figure 5. SEM images of an unloaded SF aerogel (A and C) and an SF aerogel loaded with ~21 wt% ibuprofen (B and D). Each set of images are for the same sample at two different magnifications.
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Figure 6. Side and top views of a dry SF aerogel and wet SF aerogel after soaking in PBS at 37 °C and pH 7.4. (A) dry aerogel, (B) aerogel after soaking for 1 minute, and (C) aerogel after soaking for 360 minutes.
Figure 7. In vitro release of pure ibuprofen (△) and ibuprofen-loaded SF aerogel (□) in wellstirred PBS at 37 °C and pH 7.4; A) 0 - 30 minutes and B) 0 - 400 minutes. Measurements are performed in triplicate.
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Highlights
• Silk fibroin aerogels are synthesized using sol-gel and supercritical CO2 technology
• Greater than 99.98 wt% of ibuprofen is in the amorphous form
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• The ibuprofen release from SF aerogels occur over a period of six-hours
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• SF aerogels are loaded with ~21 wt% of ibuprofen using supercritical CO2
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• Release of ibuprofen from SF aerogels is governed by Fickian diffusion
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S0896-8446(14)00109-0 http://dx.doi.org/doi:10.1016/j.supflu.2014.04.014 SUPFLU 2962
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
26-2-2014 23-4-2014 24-4-2014
Please cite this article as: M.A. Marin, R.R. Mallepally, M.A. M, Silk fibroin aerogels for drug delivery applications, The Journal of Supercritical Fluids (2014), http://dx.doi.org/10.1016/j.supflu.2014.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Applications
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Silk Fibroin Aerogels for Drug Delivery
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Michael A. Marin1, Rajendar R. Mallepally,*1, and Mark A. McHugh1,2 Department of Chemical and Life Science Engineering, Virginia Commonwealth University,
Materials Sciences Division, VCU Reanimation Engineering Science Center, Virginia
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2
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Richmond, VA 23284.
Commonwealth University Medical Center, Richmond, VA 23298.
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ABSTRACT: Silk fibroin (SF) is a natural protein, derived from the Bombyx mori silkworm. Silk fibroin based porous materials are being extensively investigated for biomedical applications,
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due to their biocompatibility and biodegradability. The results presented here demonstrate the potential of SF aerogels as drug delivery devices for the extended release of ibuprofen, a candidate drug. SF aerogels are loaded with of ibuprofen using supercritical carbon dioxide (scCO2) at 40 °C and 100 bar. Differential scanning calorimetry of the ibuprofen-loaded SF aerogels indicates that the ibuprofen is amorphous. Scanning electron microscopy and nitrogen adsorption/desorption analysis are used to investigate the morphology and textural properties. Phosphate buffer solution (PBS) soaking studies, at 37 °C and pH 7.4, reveal that SF aerogels do not swell nor exhibit any weight loss for up to six hours, the lifetime of the release measurements performed in the present study. In vitro ibuprofen release in PBS, at 37 °C and pH 7.4, occurs over a six-hour period when the ibuprofen is loaded in SF aerogel discs that are 1.4 cm in
1 1 of 35 Page
diameter and 0.85 cm in height. In contrast, the dissolution of the same amount of pure ibuprofen occurs in 15 minutes. Furthermore, the release of ibuprofen from these SF aerogel discs are modeled using the Ritger-Peppas model which indicates that ibuprofen release follows Fickian
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diffusion.
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KEYWORDS: Silk fibroin, Drug loading, Drug delivery, Protein hydrogel, Protein aerogel,
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Supercritical impregnation 1. Introduction
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Hydrogels are three-dimensional hydrophilic polymer networks that are being extensively investigated for biomedical applications such as tissue regeneration matrices [1, 2], cell scaffolds
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[3, 4], and drug delivery devices [5, 6]. These gels exhibit unique properties such as an open-
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pore network, structure tunability, and biocompatibility [6]. However, most hydrogels lack
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mechanical strength, which can be a problem during handling [7]. This low mechanical strength and subsequent handling issue can often be overcome by air drying the hydrogel to create a
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xerogel, freeze drying the hydrogel to create a cryogel, or drying the aerogel with supercritical carbon dioxide (scCO2) to create an aerogel. Air drying is the least complex method, but with this method the open-pore network of the hydrogel structure collapses due to capillary forces from evaporating water [8]. In contrast, freeze drying preserves the open-pore, hydrogel network by eliminating capillary forces as water is removed from the matrix by sublimation. However, the formation of ice crystals can destroy fine pore structure [9]. The drawbacks associated with both air drying and freeze drying are eliminated by using scCO2 drying, thus preserving the initial hydrogel structure.
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Once the aerogel is synthesized, several methods are available for loading a drug into the aerogel, including soaking the aerogel in a liquid solution or contacting the aerogel with a gas or supercritical fluid saturated with the drug [10, 11]. Loading the aerogel via soaking makes it
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difficult to fill small diameter pores with a liquid phase due to surface tension limitations and once loaded, an additional drying step is required. As an alternative, loading the aerogel via a
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saturated gas eliminates both the surface tension limitations and the drying step, but this type of
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processing can still be problematic due to the low drug solubility in a gas phase. A supercritical fluid (SCF) solvent can overcome many of the limitations associated with liquid and gas phase
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loading methods since an SCF solvent has a very low viscosity and a low solid-SCF surface tension, which means that SCF solvent, saturated with drug, can easily penetrate small pores. In
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that observed with a gaseous solvent [12].
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addition, an SCF solvent can have a drug solubility level several orders of magnitude higher than
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A fair amount of research has been reported on the loading of silica aerogels using supercritical fluid solvents [13-15]. However, these siliceous materials lack biodegradability,
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which is a primary prerequisite for drug delivery applications. Researchers have overcome this disadvantage by creating aerogel drug delivery devices consisting of polysaccharides [16-24] or covalently-linked proteins [9]. In this work, silk fibroin (SF) protein, from the cocoon of a Bombyx mori silkworm, is used to synthesize an aerogel for use as a drug delivery carrier. Although researchers have previously synthesized certain types of SF drug delivery matrices [18], to the best of our knowledge, there are no prior reports specifically on the synthesis and use of SF aerogels as a drug delivery devices. In the present study scCO2 is used to load ibuprofen, a candidate drug compound, into the SF aerogels. Scanning electron microscopy and N2 adsorption/desorption are used to determine
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the micro and macro structure, surface area, pore size, and pore volume of the SF aerogels. Phosphate buffer solution (PBS) soaking studies are performed to determine the amount of aerogel volume change and weight loss. The release rate of ibuprofen from the SF aerogels is
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measured and contrasted with the dissolution rate of pure ibuprofen to ascertain the impact of
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aerogel morphology on the delivery of ibuprofen.
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2. Materials and Methods
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2.1 Materials
Bombyx mori silkworm cocoons are purchased from Aurora silk (Portland, Oregon,
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USA). Sodium carbonate, ibuprofen, and lithium bromide are purchased from Sigma Aldrich,
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USA. All solvents are analytical grade and used without further purification.
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2.2 Preparation of silk fibroin solution and aerogels Cocoons are treated with an aqueous solution of 0.02 M Na2CO3 (1% w/v) at 90 °C for 60 minutes. Degummed fibers are recovered and washed with boiling water to remove the sericin protein that coats the SF protein. These fibers are stretched and dried overnight at 40 °C and are dissolved in 9.3 M aqueous LiBr solution (10% w/v) at 65 °C for four hours. The fiber-LiBr aqueous solution is cooled to 25 °C and dialyzed (Snakeskin Dialysis Tubing, Thermo Scientific, USA, MWCO 3,500 Da) against distilled water for three days (2.5% v/v), with every day water replacement. An aqueous 4 wt% SF solution is poured into a 10 mL glass beaker that is placed in a 300 mL stainless steel vessel (Parr Instruments, USA), which is heated to 40 °C and pressurized
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overnight with CO2 at 100 bar. The stainless steel vessel is slowly depressurized over 1.5 hour period and the recovered SF hydrogel is converted into an alcogel by soaking in aqueous solutions of increasing alcohol concentration (20, 40, 60, and 80 wt%) for 30 minutes each,
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followed by soaking in pure ethanol overnight. Ethanol is removed from the SF alcogel using scCO2 at 40 °C and 100 bar to create the SF aerogel. The volume of the SF aerogel is ~10-to-
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20% smaller than the SF hydrogel. It is worth noting that no attempt is made to optimize the
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processing of the SF aerogel, which should be investigated in the future in a similar way as done
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by Della et al. [25].
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2.3 Ibuprofen loading in silk fibroin aerogels by supercritical CO2
Ibuprofen is loaded into the aerogel using scCO2 following the method reported by Shen
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et al. 2008[26]. SF aerogel is first dehydrated at 100 °C under vacuum for two hours at -762 mm
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Hg and then placed into an 80 mesh stainless steel basket that is attached to an impeller rod
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located in a stainless steel vessel. Excess solid ibuprofen is placed in the bottom of the vessel so that scCO2 remains saturated during this loading process, which is run for two hours at 100 bar at 40 °C with stirring. Ibuprofen is expected to partition between the CO2-rich phase and the SFrich phase [26, 27]. The loading of ibuprofen, 20.6 ± 4.1 wt% (mean ± standard deviation, n = 4), is determined gravimetrically and is cross checked using TGA as subsequently described.
2.4 Silk fibroin aerogel characterization Aerogel morphology is determined using scanning electron microscopy (SEM) (HITACHI SU-70). The aerogels are lightly dabbed on double sided carbon tape stuck to
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aluminum stubs, then coated with ~10 nm of platinum via spin coating (Denton Vacuum, USA, Model: Desk V TSC). Aerogel surface area, pore size, and pore volume are determined using nitrogen
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adsorption/desorption measurements at 77 K (Quantachrome Instruments, Nova 2200e).
Approximately 50 mg of aerogel is heated at 120 °C under vacuum for two hours to dehydrate
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the aerogel before performing the adsorption/desorption measurements. The specific surface
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area of the aerogel is calculated with the multipoint Brunauer–Emmett–Teller (BET) model in the relative pressure range of 0.05 to 0.30, and the pore size distribution is calculated
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with the Barrett-Joyner-Halenda (BJH) model using a desorption isotherm.
Ibuprofen loading in the aerogel is determined using Thermal Gravimetric Analysis
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(TGA) (Perkin–Elmer USA, Model Pyris 1 TGA). The furnace is continuously flushed with
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nitrogen gas at a flow of 3 L/hour. The aerogels are rapidly heated to 80 °C, held at this
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temperature for 15 minutes, and then rapidly heated to 200 °C and held for 30 minutes. In both cases the heating rate is 100 °C/minute.
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The physical state of the ibuprofen in the aerogel is determined using differential scanning calorimetry (TA instruments, Q10). The aerogel sample is heated at 10 °C/minute from 40 to 135 °C. Sample purge flow is set to 50 mL/minute, and resultant data from the first heat are analyzed using Universal Analysis 2000 software.
2.5 Silk fibroin aerogel soaking studies The volume change of an SF aerogel disk is determined by noting the change in the length and diameter of the disk submerged in 50 mL of well-stirred PBS at 37 °C and pH 7.4. Before immersion in PBS, the aerogel is dried at 100 °C under vacuum for two hours. The SF
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aerogel is removed from the solution for immediate imaging and dimension measurement after one minute and 360 minutes. It is worth noting that macroscopic ibuprofen crystals are readily apparent to the naked eye when the loading is increased above ~60 wt%. When ibuprofen is
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loaded “in” the aerogel the ibuprofen is amorphous due to interactions with the fibroin matrix. Once the internal surfaces are saturated with ibuprofen, the excess ibuprofen precipitates as a
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crystalline solid since there are no more sites available to inhibit crystallization. Several literature
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studies have reported similar observations and interpretations on the solid form of ibuprofen or
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alkyl ketene dimer in matrices [27-29].
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2.6 In vitro drug release study
A known amount of ibuprofen-loaded SF aerogel or pure ibuprofen is put into a stainless
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steel basket made with 80 mesh screen. The basket is fully immersed in 50 mL of well-stirred
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PBS at 37 °C and pH 7.4. Perfect sink conditions are maintained during the release experiments
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since the 50 ml of PBS remain below 10% of the 6.02 mg/mL maximum solubility of ibuprofen [30]. Aliquots of PBS are removed at specific time intervals to quantify the release rate of ibuprofen and the same amount of fresh PBS at 37 °C is added back to the parent solution to maintain perfect sink conditions. The ibuprofen solution concentration is quantified using UVVis spectroscopy from the absorbance at 264 nm.
3. Results and Discussion 3.1 Silk fibroin aerogel characterization
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Figure 1 shows the surface area, pore size, and pore volume of the SF aerogels synthesized in this study as determined by adsorption/desorption isotherms. The isotherm exhibits type IV behavior and shows a significant hysteresis indicating the presence of
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mesopores. Figure 2 shows the pore size distribution of a representative SF aerogel which ranges from ~5 to 130 nm. The SF aerogels synthesized in this study have surface areas of 424 ± 75
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m2/g and densities of 0.058 ± 0.001 g/mL (mean ± standard deviation, n = 3). These SF aerogels
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have textural properties very similar to those exhibited by other whey protein aerogels [9].
DSC is used to probe the physical state of ibuprofen in the SF aerogel matrix. Figure 3
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shows DSC curves for pure ibuprofen, pure SF aerogel, and ibuprofen-loaded SF aerogel. The pure ibuprofen curve in Figure 3A has a sharp endothermic peak at ~77 °C, which is from the
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melting of crystalline ibuprofen. The pure SF aerogel curve in Figure 3B does not contain any
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peaks indicating that the unloaded aerogel does not have any crystallinity. However, the curve
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for the SF aerogel loaded with ~19 wt% ibuprofen exhibits a small endothermic peak at ~77 °C, indicating that more than 99.98 wt% of the loaded ibuprofen is amorphous. Evidently the interactions between ibuprofen and the SF aerogel matrix effectively inhibit the crystallization of ibuprofen. The results obtained in this study are consistent with the results reported by Kazarian et al. who loaded up to 30 wt% of amorphous ibuprofen into poly(vinyl pyrrolidone) using scCO2 assisted impregnation [27].
Figure 4 shows the weight loss curves for pure ibuprofen, SF aerogel, and ibuprofenloaded SF aerogel obtained by TGA. The dashed line shows the temperature profile which includes a 15 minute isothermal step at 80 °C to evaporate any water, followed by a 30 minute
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isothermal step at 200 °C to decompose and evaporate ibuprofen. SF aerogels synthesized in this study typically have 4-5 wt% of water that evaporates at 80 °C, and another 1-2 wt% of SF weight loss during the remainder of the TGA run. Figure 4 also shows that pure ibuprofen
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decomposes and evaporates rapidly at 200 °C and that the ibuprofen-loaded SF aerogel loses ~25 wt% during the 200 °C isothermal step. This weight loss is exactly matches the amount of loaded
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ibuprofen determined gravimetrically.
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The micro and nano structures of the SF aerogels are investigated by analyzing SEM images of representative SF aerogels and ibuprofen-loaded SF aerogels. The SEM images in
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Figure 5 (A-D) show the nanofibrous network of the loaded and unloaded aerogels. This fibrous network is stabilized by crystalline beta sheet conformations formed from inter- and
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intramolecular hydrogen bonding and hydrophobic interactions [31]. The crystalline beta sheets
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are formed during the sol-gel transition of silk fibroin as determined from a Fourier transform
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infrared spectroscopy analysis not shown here. There is a strong positive correlation between the pore sizes observed in the SEM images and the pore size distribution data obtained from the N2 adsorption/desorption analysis shown in Figure 2. It is worth noting that macroscopic ibuprofen crystals are readily apparent to the naked eye when the loading is increased above ~60 wt%. When ibuprofen is loaded “in” the aerogel the ibuprofen is amorphous due to interactions with the fibroin matrix. Once the internal surfaces are saturated with ibuprofen, the excess ibuprofen precipitates as a crystalline solid since there are no more sites available to inhibit crystallization. Several literature studies have reported similar observations and interpretations on the solid form of ibuprofen or alkyl ketene dimer in matrices [27-29]. No attempt is made to determine the
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saturation solubility of ibuprofen in the aerogel, above which pure ibuprofen crystals are
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observed.
3.2 Silk fibroin aerogel soaking studies
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Figure 6 shows images and dimensions of the SF aerogels after soaking in PBS at 37 °C ratio of ~1.75, whereas after one minute in
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and pH 7.4. Initially the SF aerogel has a
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solution the ratio reduces ~1.65 where it remains for the next six hours. Although the
ratio remains constant during the PBS soaking studies, the aerogel has already shrunk by ~46 %
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of its initial volume. The aerogel shrinkage is likely due to a combination of effects. Shrinkage can occur due to attractive ionic interactions between de-protonated carboxylic acid groups and
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protonated amino groups. Shrinkage can also occur due to the collapse of macropores by capillary forces exerted from rapid water transport into the matrix. It is likely that both
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phenomenon occur simultaneously resulting in matrix collapse. Further studies are in progress to elucidate the cause behind this aerogel shrinkage. It is worth noting that gravimetric analysis of the SF aerogel after the soaking experiment reveals no aerogel weight loss. Therefore, the release of ibuprofen is likely only based on diffusion from the matrix since dimensional changes in the SF aerogel matrix are not observed.
3.3 In vitro drug release studies In vitro ibuprofen release studies are performed at perfect sink conditions to ensure there are no concentration effects on the diffusion and dissolution of ibuprofen. Figure 7 shows the release profile for pure ibuprofen and ibuprofen-loaded SF aerogels in PBS at 37 °C and pH 7.4.
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Pure ibuprofen is completely dissolved within 15 minutes, whereas ibuprofen from the SF aerogel is released over a period of approximately 360 minutes. Note that the ibuprofen loaded in the SF aerogels exhibits a release profile composed of two different rates. Initially, ~75 wt% of
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the ibuprofen is released in 100 minutes followed by a significantly slower release of ~15 wt% of the ibuprofen from 100 to 360 minutes. The remaining 10 wt% of the ibuprofen is either strongly
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bound to the SF aerogel matrix or is trapped in collapsed pores in the matrix and would not be
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released until the aerogel degraded. The ibuprofen-loaded SF aerogel release profiles are similar
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to those observed for ibuprofen loaded alginate and eurylon starch aerogels [16].
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Ibuprofen release behavior from the silk fibroin aerogels is analyzed using the Ritger-
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Peppas model, which is given by:
Mt kt n M
(1)
where Mt/M∞ is the fractional weight release, k is a constant, t is time, and n is the diffusional exponent that depends on the release mechanism [32]. This model is valid for the first 60 wt% of total drug release and the model assumes no swelling of the drug delivery device, which is an accurate assumption given that the dimensions of the SF aerogel remains constant one minute after immersion in PBS. The diffusional exponent in equation 1 is determined by minimizing the mean average percent deviation (MAPD) between predicted and experimental release of ibuprofen from the SF aerogel. The MAPD is given by:
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1 n Experimental releasei Predicted releasei 100 n i1 Expermental releasei
(2)
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MAPD
where n is equal to the number of data points. A diffusional exponent of 0.43 ± 0.03 (mean ±
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standard deviation, n = 3) and an MAPD of 1.1 ± 0.3 % (mean ± standard deviation, n = 3) is
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obtained when fitting the data to equation 1. This value of the diffusional exponent obtained for the release of ibuprofen from the disk shaped SF aerogels suggests that the diffusion is Fickian
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[32].
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4. CONCLUSIONS
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The high surface area and open pore network of SF aerogels, along with their
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biocompatibility and biodegradability [33], make them attractive drug delivery devices. The present work demonstrates the ability of silk fibroin aerogels to deliver a candidate drug for an
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extended period of time. Ibuprofen loading using supercritical CO2 resulted in SF aerogels loaded with ~21 wt% of ibuprofen. Release of ibuprofen from SF aerogels is found to be governed by Fickian diffusion. Further studies are in progress to demonstrate the effect of tailoring the morphological and textural properties of SF aerogels to attain a drug delivery device that yields different target drug release profiles.
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AUTHOR INFORMATION Corresponding Author
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* E-mail: [email protected]
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge partial support for this work from the Office of Naval Research from Grant N000140710526. We also express thanks to the reviewers for their
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scientific inputs.
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Figure 1 . Typical N2 adsorption (●)/desorption (○) isotherm for the SF aerogels synthesized in this study.
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Figure 2. Typical pore size distribution of SF aerogels synthesized in this study.
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Figure 3. Differential scanning calorimetry curves. (A) pure ibuprofen and (B) pure SF aerogel (dashed line) and ibuprofen-loaded SF aerogel (solid line) obtained in this study. All measurements are performed in an open pan.
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Figure 4. Weight loss curves for pure ibuprofen (△), SF aerogel (○), and ibuprofen-loaded SF aerogel (□). The dashed line represents the TGA temperature profile.
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Figure 5. SEM images of an unloaded SF aerogel (A and C) and an SF aerogel loaded with ~21 wt% ibuprofen (B and D). Each set of images are for the same sample at two different magnifications.
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Figure 6. Side and top views of a dry SF aerogel and wet SF aerogel after soaking in PBS at 37 °C and pH 7.4. (A) dry aerogel, (B) aerogel after soaking for 1 minute, and (C) aerogel after soaking for 360 minutes.
Figure 7. In vitro release of pure ibuprofen (△) and ibuprofen-loaded SF aerogel (□) in wellstirred PBS at 37 °C and pH 7.4; A) 0 - 30 minutes and B) 0 - 400 minutes. Measurements are performed in triplicate.
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Highlights
• Silk fibroin aerogels are synthesized using sol-gel and supercritical CO2 technology
• Greater than 99.98 wt% of ibuprofen is in the amorphous form
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• The ibuprofen release from SF aerogels occur over a period of six-hours
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• SF aerogels are loaded with ~21 wt% of ibuprofen using supercritical CO2
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• Release of ibuprofen from SF aerogels is governed by Fickian diffusion
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*Graphical Abstract (for review)
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Figure 3B
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Figure 7B
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