silk fibroin ( sf ) is a natural polymer produced by a variety of insects and spiders .
the best characterized silks are the dragline silk from the spider nephila clavipes and the cocoon silk from the domesticated silkworm bombyx mori , which has been used in textile production clinical sutures , and more recently as a scaffold for tissue regeneration [ 13 ] .
bombyx mori silk is composed of a filament core protein , silk fibroin , and a glue - like coating consisting of a nonfilamentous protein , sericin . sf is characterized by repetitive hydrophobic and hydrophilic peptide sequences   and consists of heavy and light chain polypeptides of ~390  kda and ~26  kda , respectively , linked by a disulfide bond at the c - terminus of the two subunits .
the primary structure of bombyx mori sf protein is characterized by the presence of three amino acids in a roughly 3  :  2  :  1 ratio : glycine ( 45% ) , alanine ( 30% ) , and serine ( 12% ) ; and the sequence is dominated by [ gly - ala - gly - ala - gly - ser]n . sf chains also contain amino acids with bulky and polar side chains , in particular tyrosine , valine , and acidic amino acids .
the repetitive sequence in hydrophobic residues dominates the -sheet structure , forming crystalline regions in sf fibers and films .
hydrophobic regions of silk fibroin in aqueous solution assemble physically by hydrophobic interactions and eventually organize into hydrogels .
silk fibroin exhibits impressive mechanical properties as well as biocompatibility making it an attractive biomaterial and scaffold for tissue engineering .
the fibroin protein is one kind of biological materials used for artificial skin and other medical applications . as a result of its biodegradability ,
sf was evaluated for several biomedical applications . in one example , sf - based films with a thickness of 10100  m were developed for acceleration of wound healing and could be peeled off without damaging the newly formed skin . as such , the application of wound protective membranes made from sf was investigated .
sf is considered a suitable material for skeletal tissue engineering because of its good oxygen and water - vapor permeability and its minimal inflammatory reaction in vivo [ 6 , 9 ] .
as reported previously , fibroin hydrogel scaffolds were prepared from sf aqueous solution with addition of 30% glycerol to promote in situ bone regeneration .
also , sf was investigated as the substratum for the culture of animal cells in place of collagen . in another application ,
the aqueous sf solution was used to prepare a membrane for immobilization of aspergillus niger , glucose - oxidase , and pseudomonas fluorescens lyophilized cells . a novel biocompatible blend  
was prepared from recombinant human - like collagen ( rhlc ) and used as a scaffold material for hepatic tissue engineering applications .
solution blending was used to incorporate rhlc with sf to enhance the blend films biocompatibility and hydrophilicity , while maintaining elasticity . in
yet another demonstration of sf utility , three - dimensional microperiodic scaffolds for tissue engineering were produced from aqueous solutions of regenerated bombyx mori silk .
the scaffolds supported human bone - marrow - derived mesenchymal stem cell ( hmsc ) adhesion and growth .
sf was studied as an organic polymer for controlled drug delivery , in which dextrans of different molecular weights , as well as proteins , were physically entrapped into the drug delivery device during processing into films .
the release behavior of benfotiamine , a vitamin b1 derivative , from sf gel was investigated .
microspheres were fabricated from an aqueous sf solution by laminar jet break - up flow and were investigated as a platform for controlled drug delivery .
the assembly process was reported for sf particles loaded with small molecule model drugs , such as alcian blue , rhodamine b , and crystal violet , produced by an all - aqueous salting out process , and it was demonstrated that the release kinetics of crystal violet is dependent on the secondary structure of the sf particles .
we attempted to design an oral drug delivery system based on the ability of sf to undergo conformational transition from a random coil to a -sheet form to induce crystallinity and produce an interpenetrating network ( ipn ) .
several different approaches to develop a sf - based drug delivery system were used : ( 1 ) film and matrix casting with varying composition of sf , gelatin , glycerin and the model drug , and ( 2 ) spray drying of sf / model drug solution .  
multiple factors were evaluated for their effect on sf -sheet formation , including solvents , sf molecular weight , silk source , and so forth .
the aim of our study is also to understand the silk fibroin processing and control of structure in connection with design of a controlled release matrix .
cocoons of bombyx mori silkworm silk were kindly provided by m. goldsmith ( university of rhode island , usa ) .
low mw ( ~14  kda ) sf powder was supplied by lalilab ( raleigh , usa ) .
raw silk fiber ( grade 5a , bombyx mori silk ) was purchased from ria international llc ( east hanover , nj , usa ) , and fibro - silk powder ( mw ~ 100  kda ) was purchased from arch chemicals , inc ( atlanta , ga , usa ) .
both sephadex g-25 ( medium grade ) and sodium carbonate were purchased from j. t. baker ( austin , tx , usa ) .
naproxen sodium was supplied by rochem international , inc ( ronkonkoma , ny , usa ) .
sodium dodecyl sulfate and calcium chloride dihydrate were purchased from spectrum chemical ( new brunswick , nj , usa ) .
lithium bromide , calcium nitrate , and potassium bromide were purchased from sigma - aldrich ( st . louis , mo , usa ) .
gelatin ( type b , 150 bloom limed bone , nf ) was obtained from rousselot ( france ) .
glycerin ( usp , kosher , vegetable - based ) was obtained from proctor and gamble ( cincinnati , oh , usa ) .
all other chemicals were of analytical or pharmaceutical grade , were purchased from sigma - aldrich , and were used without any additional purification .
silk fibroin aqueous stock solutions were prepared as described previously with some modifications [ 16 , 18 ] .
briefly , cocoons , silk powder , or grade 5a raw silk were boiled several times for 1 hour in aqueous solutions of 0.02  m na2co3 , or 0.25% naco3/0.25% naso4 mixture , rinsed thoroughly with distilled water to remove the glue - like sericin proteins and dried .
dry degummed silk fibers were then dissolved in one of the following neutral solutions of libr , ca(no3)2 or cacl2 , and then the resulting solution was dialyzed against distilled water using a slide - a - lyzer dialysis cassette ( mwco 3500 , pierce ) or cellulose membrane tube ( mwco 60008000 , sigma - aldrich ) at room temperature to remove the salt .
the final concentration of sf aqueous solution was determined by weighing the residual solid of a known volume of solution after drying at 60c for 2 days .
based on this determination , the concentration of the silk protein was approximately in the range of 3 to 4% ( w / v ) . to prepare films ,
sf solution was transferred to a polystyrene weighing boat and allowed to dry for several days at room temperature in a desiccator .
sf / gelatin films were prepared by mixing the sf solution with gelatin blends , consisting of gelatin , plasticizer , and water , and dried in a polystyrene weighing boat at room temperature in a desiccator for several days .
separation of salts and sf protein was performed using a sephadex g-25 media column as described in the literature   with some modifications . sf powder was dissolved in a triad solvent of cacl2  :  etoh  :  h2o with a mole ratio of 1  :  2  :  8 , at a concentration of 14.4% ( w / w ) , at 6080c , and stirred for 46  hrs until fully dissolved and the stock sf solution was diluted in deionized water to reduce sample viscosity . to a 7.3  g of sephadex g-25 ( medium grade ) 42.6  g water was added allowing the sephadex to swell for at least 3 hours then the slurry was packed by gravity flow of deionized water ( 2 - 3 bed volumes ) in a 50  ml glass burette .
conductivity of eluent flow was measured until 3 consecutive fractions ( 10  ml each ) tested < 10  s / cm to ensure removal of contaminating ions from column before addition of sf solution ( 7.2% sf ) .
fractions were collected every 510 minutes for the first ~25 minutes , while conductivity was continuously measured then every 25 minutes until the end of the experiment , or until the conductivity of the eluting fraction returned to a value of < 10  s / cm .
uv absorbance was measured and recorded for each fraction at 280  nm ( blank  : quartz cuvette filled with deionized water ) .
all fractions were placed in the oven at 60c for 24 hours , or until all liquid had evaporated , and the residual net mass was determined for each fraction after drying . to prepare sf microparticles , the model drug naproxen sodium ( ns ) , was dissolved in sf solution ( silk  :  naproxen ratios tested : 1  :  1 , 1.5  :  1 , 2  :  1 , and 3  :  1 ) for spray - drying .
naproxen - sodium - containing sf microparticles were prepared using a bench top spray - dryer ( bchi b-290 model , switzerland ) .
the adjustable parameters included inlet and outlet temperature , solution pump flow rate , and the aspirator partial vacuum . in the present experiments , the inlet temperature ranged from 135 to 200c ,
the pump flow rate was 6 - 7  ml / min , and the atomizing air flow rate was 600  l / h ( 0.75  bar ) .
the morphological characteristics of sf microparticles and cross - sections of gelatin / sf blends , both before and after treatment with dehydrating solvents and before and after drug release experiments , were examined by scanning electron microscopy ( sem ) .
samples of dried films were prepared for sem imaging by flash freezing in liquid nitrogen and immediately fracturing with a sharp blade to produce cross - sections .
the samples were then coated with a ~8  nm layer of au / pt mist using a sputter coater . a hitachi s3200 variable pressure scanning electron microscope equipped with an e - t secondary electron detector
was used to examine the samples at an accelerating voltage of 5  kv and a working distance of 15  mm .
particle size measurements for silk microparticles were also performed using dynamic light scattering ( dls ) . a nicomp zw380 ( particle sizing systems , inc .
ca ) particle sizer at fixed angle ( 90 ) and 632.8  nm was employed for size distribution measurements .
the sf microparticles were suspended in an oil matrix at a 1  :  100 dilution before measurements .
ftir spectra of silk fibroin blend films were collected using a nicolet 510p spectrometer equipped with the attenuated total reflectance ( atr ) accessory .
each spectrum for samples was acquired in transmittance mode with a spectral range of 4000400  cm .
ftir spectra of the sf - containing microparticles were collected using a perkin - elmer spectrum bx ftir spectrophotometer and nic380 avatar omni sampler apparatus .
kbr pellets , containing ~0.7% ( w / w ) of spray - dried powder were prepared using a carver pellet press with 18,000 lbs applied load .
thin films for comparison were obtained by casting of sf solution on the surface of polystyrene weigh boats and were analyzed by ftir . the naproxen release from the films , matrixes , and spray - dried microparticles
two sets of conditions were used : one - stage method with apparatus 2 ( paddle ) at 50  rpm in 500  ml of ph 7.4 phosphate buffer at 37c and three - stage method with three ph buffers  simulated gastric fluid ( ph 1.2 ) , simulated intestinal fluid ( ph 4.8 ) and ph 7.4 buffer .
the samples ( 1.5  ml ) at each predetermined time point were analyzed by hplc according to naproxen sodium , usp method .
the detector wavelength was set at 272  nm and the injection volume was 10  l .
the hplc column used was inertsil ods-3 4.6  250  mm , 5  m with the column temperature maintained at 25c .
the mobile phase was a binary mixture of potassium phosphate buffer  :  acetonitrile ( 3  :  2 ) at ph 3.0 pumped at flow rate of 1.5  ml / min .
raw silk consists of fibroin that is bound together by hydrophilic gum - like protein , the sericin . for use in biomedical applications ,
the dissolution of sf as related to the roles of salts , alcohol , and water and coagulation of the fibroin solution was discussed elsewhere .
lithium bromide  or lithium thiocyanate  ethanol system , hexafluoroisopropyl alcohol , and calcium nitrate - methanol systems have been widely used to dissolve silk fibroin .
a summary of the processing conditions employed for different sources of sf in our research is presented in table 1 .
it usually takes longer time ( 7 - 8 hours ) at 6580c to dissolve raw bombyx mori silk using different solvent systems ( table 1 ) , then to dissolve sf powder ( 46 hours ) . in order to develop a scalable process we have attempted to separate sf from salts by sephadex g-25 media as described in the literature .
the primary purpose of purifying by column chromatography was to explore the feasibility of a quicker processing step , in place of dialysis .
the two components were effectively separated using this approach , allowing for gravimetric analysis as a means of approximating the mass of either sf protein or cacl2 salt contained in each fraction collected ( figure 1 ) .
these results demonstrate the feasibility of separating sf protein dissolved in a highly concentrated salt solution ; however , further characterization of the sf protein after desalting will be required prior to implementation of this process on a commercial scale . in order to reduce the processing time we explored the applicability of different sources of silk fibroin .
one option was to eliminate the degumming step and use partially hydrolyzed sf , while another option was to use a low - molecular - weight water - soluble sf , thus allowing elimination of three processing steps : degumming , dissolving , and dialysis .
the ability to form crystalline structure was investigated for partially hydrolyzed sf at three different molecular weights ( 100  kda , 14  kda , and 2  kda ) .
low mw sf ( 14 and 2  kda ) was soluble in water and demonstrated crystalline secondary structure in pure form as evidenced by ftir data presented in table 2 .
however , only higher - molecular - weight sf ( 100  kda ) was shown to possess -sheet conformation in blends with gelatin .
the effect of glycerin and dehydrating solvents ( methanol , ethanol , and isopropyl alcohol ) on formation of -sheets was studied for gelatin / silk fibroin compositions .
sf / gelatin compositions with and without glycerin were prepared and treated with methanol , ethanol , and isopropyl alcohol . the effect of different solvents and presence of glycerin on -sheet formation is summarized in table 3 .
the effect of solvents on -sheet formation showed that films treated with different alcohols exhibit the signature of the -sheet conformation ( silk ii structure ) at each amide peak : amide i , which reflects the stretching of c = o group along the sf backbone ( shifted from ~1650  cm to 1630  cm ) .
the amide ii , which originates from n  h deformation , shifts from ~1544 to 1536  cm .
higher sf ( sf / g 1  :  1 ) content showed higher fibroin - specific signal change for amide i after cast - treatment .
interestingly , the untreated films ( sf / g 1  :  1 ) also demonstrated the crystallizing effect indicating that glycerin could induce the formation of -sheet ( table 3 ) .
a mixture of dialyzed sf solution with predetermined amount of gelatin mass and a model drug was cast on a polystyrene weighing boat to prepare sf films .
cast films were treated with methanol , ethanol , and isopropyl alcohol or exposed to water vapor .
gelatin mass was prepared from gelatin , water , and plasticizer ( glycerin ) by initially mixing water and plasticizer with gelatin granules followed by heating at ~60c until a clear gel was obtained .
sf - containing compositions were prepared , using naproxen sodium , as a model drug and presented in table 4 .
these compositions are calculated based on weight after the films and matrixes have completely dried , before performing dissolution testing .
the characterization of naproxen release from sf - containing matrixes and films was performed at ph 7.4 .
drug release from amorphous carrier ( control film ) was characterized by an initial burst exceeding 75% of the theoretical amount of naproxen in 5 minutes demonstrating immediate release of the model drug .
for sf - containing films the initial burst was markedly reduced ( ~60% in 5 minutes ) .
studies ( figure 2 ) indicated that the time needed to achieve over 80% dissolution for naproxen - loaded films is 15 minutes as opposed to 5 hours for the sf - containing matrix .
these results demonstrate the formation of crystalline sf network in silk and gelatin blends which significantly retard the release of naproxen compared to amorphous gelatin .
although the sf / gelatin / glycerin blends described above demonstrated feasibility for use as a controlled drug delivery system , another approach utilizing microparticles containing only sf and water was explored .
sf - containing compositions loaded with naproxen sodium were prepared via spray - drying and are presented in table 5 . the spray - dried powders with different ratios of sf  :  ns ( 1  :  1 , 1.5  :  1 , 2  :  1 and 3  :  1 ) were encapsulated in 2-piece hard gelatin capsules , and the release of naproxen sodium was studied at ph 7.4 .
the dissolution study results were compared to the release profile of naproxen - loaded sf / gelatin thin films . as shown in figure 3 sf films released 90% of naproxen sodium within 1 hour of dissolution time at ph 7.4 , while sf spray - dried powder released only ~50% of naproxen sodium .
approximately 4 hours were required for sf spray - dried powder to release above 80% of naproxen sodium .
the controlled release observed for naproxen from the microparticles compared to the fast release rate from the film suggests that the spray - drying process induces a unique change to the structure of the fibroin microparticles thereby transforming them into prospective sustained release vehicles .
this provided a starting point for developing sf spray - dried microparticles as a drug delivery system .
the release of naproxen sodium from spray - dried microparticles was also demonstrated using a three - stage dissolution method at ph 1.4 , 4.5 and 7.4 .
the microparticles with sf : ns ratios of 1.5  :  1 , 2  :  1 , and 3  :  1 were observed to have similar release profiles while microparticles with sf  :  ns ratio of 1  :  1 had slightly higher release profiles . at stage 3
( ph 7.4 ) dissolution , microparticles with different sf  :  ns ratios performed similarly , releasing up to 80% ns after 3 hours ( figure 4 ) , while at stage 2 ( ph 4.5 ) the release of the drug was in the range of 1020% .  
as seen in figure 4 , < 25% of drug release from sf microspheres is observed over the first 6 hours at or below ph 4.5 , even at the lowest ratio of sf  :  ns ( 1  :  1 ) .
complete drug release is only observed after 15 hours at ph 7.4 ( 25 hours total dissolution time ) for all samples analyzed .
ftir analysis was performed on spray - dried microparticles , and the data showed that spray - drying of sf solution induced -sheet conformation which was indicated by the amide i band shifting from 1650  cm to 1642/1631  cm and amide ii from 1536  cm to 1516  cm .
however , most of the analyzed spray - dried microparticles did not show -sheet transition .
samples exposed to 76% relative humidity for one week showed -sheet transition as evidenced by the shift of amide i , ii , and iii bands ( table 6 ) , thus demonstrating that exposure to humidity induced -sheet formation . to further investigate the effect of water vapor treatment on conformation change we subjected the sf  :  ns spray - dry powder exposed to 76% relative humidity to 3-stage dissolution studies .
the slight changes in dissolution may suggests that exposure to moisture contributed to conformational transition in sf spray - dried powder .
sf  :  ns ( 2  :  1 ) spray dried powder exposed to 76% relative humidity provides similar release during stage 1 and 2 dissolution testing , but showed slightly lower ns release during stage 3 testing . these data
could support the hypothesis that the conformational transition rate depended on the rearrangement of hydrogen bonds between sf chains ; however additional studies are needed for confirm this assumption .
previous work has shown that the conformation transition of sf from random coil to -sheet is due to the rearrangement of the hydrogen bonds between the polypeptide chains and the transition may be a nucleation - dependent aggregation .
the modest effect on dissolution performance observed ( figure 5 ) was due to the fact that when both the sf microparticles that were initially exposed to water and those not exposed to water are subjected to the dissolution media , they can further rearrange their chains making the initial humidity effect negligible .
the data presented in figure 5 demonstrated that the release profile of naproxen from sf microparticles exposed to either high humidity ( 76% ) or 60c for 1 week is comparable to naproxen release from control samples .
sem images 6(a ) , 6(b ) , 6(c ) , and 6(d ) show the microparticles formed from each spray - dried powder blend .
the images provide evidence that microparticles were formed during the spray drying process . as seen in the images the sf microparticles collapsed , leaving behind imploded micro - particulates .
the addition of 5% ethanol to microparticles loaded with naproxen and control blends ( refered to as ( c ) and ( d ) in figure 6 ) was shown to produce microparticles that are less aggregated and more smooth , on average , which is possibly attributed to an accelerated evaporation process in the presence of alcohol .
further studies of spray - drying conditions are required to address the observation of collapsed sf microparticles . the volume , intensity , and number weighted analysis with the dls instrument showed that the mean particle size for the sf  :  ns ( 2  : 
this is in close agreement with the particle size established by the sem images .  
a series of sem images ( figure 7 ) were acquired for different spray - dried microparticles remaining after 3-stage dissolution experiments at ph 1.4 , 4.5 and 7.4 .
the microparticles were retrieved from the dissolution vessel after the endpoint at ph 7.4 and dried before sem analysis .
sem images 7(a ) , and 7(b ) represent dried sf samples that remained as a compact porous matrix .
natural silk fibers dissolve only in a limited number of solvents , compared to globular proteins , because of the presence in fibroin of a large amount of intra- and intermolecular hydrogen bonds and its high crystallinity and specific physicochemical properties .
the isoelectric point of fibroin varies in the range ph 3.65.2 , depending on the conditions of solution preparation .
fibroin dissolves in concentrated aqueous solutions of acids ( phosphoric , formic , sulfuric , and hydrochloric ) and in concentrated aqueous , organic , and aqueous - organic solutions of salts [ licns , libr , cacl2 , ca(cns)2 , zncl2 , nh4cns , cuso4 + nh4oh , ca(no3)2 ] .
the main disadvantages of salt - containing aqueous , aqueous - organic , and organic solutions of fibroin are the long preparation time ( aqueous solutions of fibroin should be dialyzed for several days ) .
it should be noted that the concentrations of salts in such solutions reach the saturation limit .
it was reported   that the efficiency of aqueous salt systems depends on the salt concentration and increases in the following order : for anions , sulfate < citrate < tartrate < acetate < chloride < nitrite < bromide < iodide < thiocyanate < dichloroacetate ; for cations , ca < sr < ba
< li < zn . a 75  :  25 ( weight ratio ) mixture of ca(no)2    4h2o and absolute methanol was used earlier for dissolving bombyx mori silk [ 24 , 25 ] as it has the strongest dissolving capacity for the sf . some solvent systems containing libr , licns , and ca(cns)2 are unfavorable because libr , licns , and ca(cns)2 are classified as toxic chemicals . hence , in this study two solvent systems cacl2  :  etoh  :  h2o ( 1  :  2  :  8 mole ratio ) and ca(no)2    4h2o were utilized for sf processing . since purification of sf by dialysis usually takes 3 - 4 days and is applicable only for small batches of sf solution , we attempted to develop a scalable process using sephadex g-25 media as described in the literature .
effective chromatographic separation of sf from salt in solution was demonstrated by the data shown in figure 1 . both uv absorbance and conductivity measurements for detecting sf and cacl2 , respectively , were quick and effective techniques for differentiating between the two solution components .
analysis of solids content upon drying provided an effective means of quantifying sample mass in each fraction collected , when combined with the latter experimental techniques to determine the composition of each fraction .
high yield ( 95% recovery of sf protein ) and high productivity ( > 98% salt removal in < 2  hrs . ) shown herein for sephadex column chromatography provide a promising alternative to conventional sf purification by dialysis .
purification of sf solutions by sephadex g-25 column chromatography could be an effective and industrially scalable chemical process .
however , further optimization and analysis need to be performed for utilizing sf in pharmaceutical development .
in addition , sephadex media can be flushed and reused , thereby reducing development costs associated with purification of sf solutions .
sf is dominated in composition by the amino acids glycine , alanine and serine which tend to form antiparallel -sheets or crystals through hydrogen bonding and hydrophobic interactions . upon gelation
many factors such as temperature , sf concentration , shear force , metallic ions , ca , ph , treatment with low dielectric constant solvents and poly(ethylene oxide ) [ 21 , 26 ] are thought to affect the conformation transition . with increase in sf
it is well known and reported in the literature   that the addition of methanol to sf induces aggregation ( dehydration ) , which drives the structural transition from random coil to -sheet .
it was demonstrated [ 28 , 29 ] that upon methanol - induced crystallization , the sf -sheet network stabilizes sf / gelatin hydrogels at elevated temperatures . the transition of regenerated sf films from random coil to -sheet has been reported   after treatment with methanol , ethanol , and 2-propanol .
it was also demonstrated   that the rate of gelation of sf was dependent upon glycerol content and/or sf content and addition of glycerol to the sf solution accelerated this rate . in our research
, we investigated the effect of dehydrating solvents ( methanol , ethanol , isopropyl alcohol , and glycerin ) on formation of -sheets in sf / gelatin blends and demonstrated that the treatment with glycerin is also effective for the transformation of silk i to ii which is in agreement with the literature data .
the presence of glycerin in the matrix can trigger -sheet induction as seen from table 3 at the ratio of sf / gelatin ~1  :  1 .
since the -sheet formation did not occur in experiments with sf - to - gelatin ratio of 1  :  3 , it is suggested that the ratio of sf to gelatin is also critical for the -sheet formation . in the presence of glycerin , for the sf / gelatin 1  :  1 blend , untreated films exhibit the absorption bands characteristic of the -sheet structure . in this case
the ftir spectra possess strong absorption bands at 1625.9  cm1621.8  cm and 1536  cm1531.9  cm , corresponding to the amide i and amide ii peaks , respectively .  
the induced crystallization of sf - containing films had an impact on the release profile of the model drug naproxen sodium as evidenced by dissolution studies performed on naproxen - sodium - loaded films .
it was shown that no burst effect was observed for matrix containing sf  :  gelatin  :  glycerin in the ratio of 1  :  3  :  3 compared to films containing gelatin alone or silk  :  gelatin ( 1  :  1.5 ) only which released almost 80% of the drug within the first 15 minutes ( figure 2 ) .
the influence of glycerin - induced sf / gelatin crystallization on structure and properties was ascertained by dissolution studies of sf containing controlled release matrixes ( figure 2 ) .
the -sheet content in the sf matrixes was assessed by ftir and illustrated in figure 8 .
two maxima on spectra reflect the characteristic bands of noncrystallized biopolymer ( gelatin ) in the matrix and crystallized sf .
the amide i peak , which reflects the stretching of c = o group along the sf backbone , is shifted from 1655 to 1630  cm , while the gelatin exhibit , the absorption band at 1654  cm ( amide i ) .
release behavior of the model drug at different loading from spray - dried microparticles was studied using 3-stage dissolution testing conditions . in our study
it was observed that the release profile was not dependant on the naproxen - to - sf ratios in the range of 3  :  1 to 1  :  1 or treatment with dehydrating solvent ( ethanol ) demonstrating that spray - drying method accelerated the transition from random coil to the -sheet structure of microparticles , which is in agreement with the literature data .
our data obtained from naproxen - loaded , spray - dried microparticles , matrices , and films demonstrated a promising approach for creating a new platform for controlled drug delivery .
it has been demonstrated that the conformational transition of sf from random coil to -sheet in blends with gelatin obtained by spray - drying or induced by solvents could be used to generate a porous matrix .
the development of sf - containing blends in which sf is crystallized yields drug delivery system allowing for controlled release of the drug .
further studies will be performed on sf - containing matrixes and microparticles to explore feasibility for delivering different classes of drugs , in particular macromolecular drugs for site - specific delivery .
 the objective of this proof - of - concept study was to develop a platform for controlled drug delivery based on silk fibroin ( sf ) and to explore the feasibility of using sf in oral drug delivery . 
 the sf - containing matrixes were prepared via spray - drying and film casting , and the release profile of the model drug naproxen sodium was evaluated . 
 attenuated total reflectance fourier transform infrared spectroscopy ( ftir ) has been used to observe conformational changes in sf- and drug - containing compositions . 
 sf - based films , spray - dried microparticles , and matrixes loaded with naproxen were prepared . 
 both ftir spectra and in vitro dissolution data demonstrated that sf -sheet conformation regulates the release profile of naproxen . 
 the controlled release characteristics of the sf - containing compositions were evaluated as a function of sf concentration , temperature , and exposure to dehydrating solvents . 
 the results suggest that sf may be an attractive polymer for use in controlled drug delivery systems . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions