Surface Modification of Silk Fibroin Fabric Using Layer-by-Layer

Subscriber access provided by NORTH DAKOTA STATE UNIV

Article

Surface Modification of Silk Fibroin Fabric Using Layerby-Layer Polyelectrolyte Deposition and Heparin Immobilization for Small Diameter Vascular Prostheses M. Fazley Elahi, Guoping GUAN, Lu Wang, Xinzhe ZHAO, Fujun WANG, and Martin W. King Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504503w • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

38x17mm (600 x 600 DPI)

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Surface Modification of Silk Fibroin Fabric Using Layer-by-Layer Polyelectrolyte Deposition and Heparin Immobilization for Small Diameter Vascular Prostheses

M. Fazley ELAHI1, Guoping GUAN1, Lu WANG1*, Xinzhe ZHAO1, Fujun WANG1, Martin W. KING1,2 1

Key Laboratory of Textile Science and Technology, Ministry of Education, College of Textiles,

Donghua University, Songjiang, Shanghai 201620, China 2

College of Textiles, North Carolina State University, Raleigh, NC 27695-8301, USA

*Correspondence to: Wang Lu (E-mail: [email protected]) Abstract There is an urgent need to develop a biologically active implantable small diameter vascular prosthesis with long term patency. Silk fibroin based small diameter vascular prosthesis is a promising candidate having higher patency rate. However, to improve its further hemocompatibility, the surface modification is indeed required. In this study, silk fibroin fabric was modified by a two stage process. First, the surface of silk fibroin fabric was coated using a layer-by-layer polyelectrolyte deposition technique by stepwise dipping the silk fibroin fabric into a solution of cationic poly(allylamine hydrochloride) (PAH) and anionic poly(acrylic acid) (PAA) solution. The dipping procedure was repeated to obtain the PAH/PAA multilayers deposited on the silk fibroin fabrics. Second, the polyelectrolytes deposited silk fibroin fabrics were treated in EDC/NHS activated low molecular weight heparin (LMWH) solution at 4 °C for


Page 2 of 34

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

24 h, resulting in immobilization of LMWH on the silk fibroin fabrics surface. SEM (scanning electron microscopy), AFM (atomic force microscopy), and EDX (energy dispersive X-ray) data revealed the accomplishment of LMWH immobilization on the polyelectrolytes deposited silk fibroin fabric surface. The higher number of PAH/PAA coating layers on the silk fibroin fabric, the more hydrophilic surface could be obtained, resulting in a higher fetal bovine serum (FBS) protein and platelets adhesion resistance properties when tested in vitro. In addition, compared with untreated sample, the surface modified silk fibroin fabrics showed negligible loss of bursting strength and thus reveals the acceptability of polyelectrolytes deposition and heparin immobilization approach for silk fibroin based small diameter vascular prostheses modification. Keywords Silk fibroin fabric, hydrophobicity, layer-by-layer deposition, heparin, contact angle, protein adsorption, platelets adhesion, small diameter vascular prostheses Introduction Protein induced hemo-incompatibility continues one of the important issues linked with the long term use of blood contacting medical devices. The necessity of understanding the influencing factors related to thrombus formation is obligatory for the progress and use of new biomaterials. It is generally established that the protein adsorption is the first incident following blood-material contact.1,2 On implantation a vascular graft, platelets first adhere on its surface via adsorbed proteins, then aggregate resulting a fibrin rich thrombus through coagulation process (Figure 1). During intrinsic pathway, coagulation process is initiated by activation of factor XII (Hageman factor, a serine protease), which then triggers the activation cascade of several other clotting factors, eventually leading to formation of factor Xa (activated Stuart-Prower factor or


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

thromboplastin) . The extrinsic system occurs just after the activation of factor VII (stable factor, serine protease) by contact with tissue factor, which becomes accessible subsequent vascular injure. Factor VIIa (activated factor VII), in turn, leads to formation of factor Xa (activated factor X) from factor X (Stuart-Prower factor or thromboplastin-an enzyme), which is the preliminary point for the common coagulation pathway. Clotting factor Xa gets contact with a cofactor, factor V (labile factor or proaccelerin), and with the incorporation of calcium (Ca) and platelet phospholipids, prothrombin is transformed into thrombin. After a while, thrombin eventually cleaves both fibrinogen and factor XIII (fibrin stabilizing factor, a transglutaminase). The fibrin monomers assemble together to form a gel like substance, which in turns converts to insoluble fibrin net with the help of and factor XIIIa (activated factor XIII). Surface activation approach to develop hemocompatibility combined with techniques to lessen both protein adsorption and platelet adhesion to the vascular graft surface thereby inhibiting coagulation process. However, all most all the modification approaches, such as bulk blending,3-5 plasma treatment,6-8 surface grafting,9-13 and coating

14-20

etc. employed to resist the formation of intimal hyperplasia and

thrombosis have failed, which are the major cause of failure small diameter vascular graft. Small-diameter vascular grafts inserted in low-flow blood vessels fail rapidly due to occlusion caused by thrombosis and fibrotic hyperplasia.21-23 Systemic anticoagulant therapy helps to maintain the patency of larger diameter (> 6 mm) vascular grafts but is not suitable for small diameter grafts. It has been confirmed through current researches that woven silk fibroin tubular fabrics offer a potential biomaterial for use as a small diameter arterial prosthesis.22,24,25 This innovative graft materials constructed from fibroin fibers exhibit outstanding patency when used in animal trial, with 1 year patency rates of 85% which is better than for ePTFE (30%).26 In our previous work, we have shown that PAH/PAA deposited and EDC/NHS treated low molecular


Page 4 of 34

Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

weight heparin immobilized silk fibroin fabrics have the ability to decrease the hemolysis rate and blood coagulation.27 To be an ideal small diameter vascular prosthesis material, others biological properties such as, resistance to protein adsorption and platelets adhesion properties need to be further investigated. Considering these requirements as an important issue, we have carried out in vitro protein and platelets adhesion assays and some other related experiments onto untreated and modified silk fibroin fabrics. In the present study we have assembled 3 and 5 polyelectrolyte layers using the layer-by-layer (LbL) by ensuring that the PAH is on the top of the layer of the modified fibroin fabric with positive charged. After that EDC/NHS activated low molecular weight negatively charged heparin (LMWH) was immobilized onto the top layer (PAH) of silk fibroin fabric. Our objective was to develop a flexible layer attached to the fibroin fabric with the ultimate goal of fabricating a flexible tubular vascular graft. To achieve this we needed to use as few layers as possible. This is because it is reported that multilayer layer-by-layer films are usually considered as nonflexible structures.28 We assume that with the deposition of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) layers and finally the immobilization of low molecular weight heparin onto the modified fibroin, we can obtain a stable and flexible heparinized surface suitable for small diameter vascular prostheses. Experimental section Materials 100% silk filament from B.mori supplied by Soho International Silk Company Ltd, Jiangsu, China was braided (warp) and woven to produce 1/1 silk fabric. Pure silk fibroin fabric was obtained by removing the natural sericin coating from the raw silk fabric by treating with 0.05%


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(w/w) Na2CO3 solution at 98°C for 90 minutes. Fetal bovine serum (FBS) and phosphate buffered saline (PBS) were purchased from Solarbio, Shanghai, China. Low molecular weight heparin (LMWH) (average Mw = 15000) (Figure S1, Supporting Information). The poly(allylamine hydrochloride) (PAH) (average Mw = 58000), and poly(acrylic acid) (PAA) (average Mw = 1800) were purchased from Sigma-Aldrich. The polyelectrolytes were used as received without further purification and were prepared as 1g/L solutions. The 1-ethyl-3(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were procured from Shanghai Medpep. Co., Ltd. Deposition of polyelectrolytes onto the silk fibroin fabric surface The basic experimental design to deposit polyelectrolytes onto silk fibroin fabric by a layer-bylayer process is shown in Figure 2. The 3 and 5 polyelectrolyte layers on the silk fibroin fabrics surfaces were generated by consecutively dipping of the substrate into aqueous solutions of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). Both PAH and PAA solutions were prepared at a concentration of 1 mg/mL by dissolving the electrolytes in distilled water (dH2O). Then the fibroin fabric was first immersed in the PAH solution and incubated at 37°C and 100 rpm for 30 min followed by rinsing 3 times, each time for 1 min in a dH2O bath. The fabric was then immersed in the PAA solution for 30 min followed by the same rinsing steps in triplicate. The electrostatic adsorption and rinsing steps were repeated until the desired number of deposition layers (3 and 5) was obtained and the outermost layer was PAH. These modified fibroin fabrics containing the polyelectrolyte bilayers were dried at room temperature for 24 h prior to the subsequent heparin immobilization.


Page 6 of 34

Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Heparin immobilization onto polyelectrolytes deposited silk fibroin fabric surface Polyelectrolytes deposited silk fibroin fabrics were immersed in EDC/NHS activated heparin solution. The concentration of the LMWH solution was fixed as 1mg/mL by dissolving the powder in PBS solution. The modified fibroin fabrics were allowed to react with the heparin solution at 4°C for 24 h. At the end of this heparinization process, the modified silk fibroin fabrics were taken out of the heparin solution and washed with PBS and then rinsed with dH2O in an ultrasonic bath for 10 min. Characterization techniques Surface morphology and roughness: The surface morphology and roughness of the untreated and modified silk fibroin fabrics was observed by scanning electron microscopy (SEM) (TM 3000, Hitachi, Japan) and atomic force microscopy (AFM) using a SPM Nanoscope IV instrument (Veeco Instruments Inc., USA). Samples were sputter coated for 5 min with gold using the fine-coat ion sputter (LDM-150D, Shanghai, China) at 50 mA and examined under SEM. EDX: A scanning electron microscope (JEOL JSM 6330, Tokyo, Japan) operating with an ISIS 300 EDX unit (Oxford Instruments, UK) with a working distance of 15 mm and beam energy of 20 Kv was used. Surface wettability: Surface wettability of the untreated and surface modified silk fibroin fabrics was measured using both captive bubble (static contact angle) and sessile drop (dynamic contact angle) methods as illustrated in the supporting information (Figure S2 and S3). Images of the water droplet as it spread over the fabric surface were captured with a Teli CCD camera with macro lens assembly and IDS Falcon/Eagle Framegrabber (DataPhysics Instrument GmbH,


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 34

Filderstadt, Germany). In addition to the contact angle tests, the surface free energy and its polar and dispersive components of the untreated and modified silk fibroin fabrics were calculated using Owen-Wendt method according to the equation (1).29 2( γ sd γ ld + γ sp γ lp ) = γ l (1 + cos θ ) , p

(1) d

where, (γ s ): polar component of surface free energy, (γ l ): dispersive component of surface free energy, ( s ) : solid substrate , ( l ) : liquid, (θ ): contact angle of liquid on a solid surface. Bursting strength: The bursting strength of untreated and modified silk fibroin fabrics was measured on a universal mechanical tester (Model: YG-B 026H) with reference to the method described in the standard ISO7198:1998. The untreated and modified silk fibroin fabrics were cut into pieces measuring 2×2 cm2 and these specimens were mounted between the two clamping plates. The clamping device was fastened to the bottom of the machine while the probe (diameter 1.5 mm) was forced through the specimen at a speed of 50 mm/min to generate the maximum breaking force. The bursting strength of each sample was then calculated according to the equation (2). F=T /πr 2 ,

(2)

where, F is the bursting strength, T is the maximum breaking force and r is the radius of the probe (0.75 mm). XRD analysis: X-ray diffraction of untreated and modified silk fibroin fabrics were recorded on an X-ray diffractometer (Rigaku D/max 2550/PC, Japan) with Cu Kα radiation (λ= 0.154 nm), operated at 40 kV and 200 mA and the scanning rate was 0.02°/min.


Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Protein adsorption assay: In order to determine whether the fibroin fabric’s affinity for blood plasma proteins changed or not as a result of the surface modification, the protein adsorption assay was performed. Before exposing to fetal bovine serum (FBS), the untreated and surface modified silk fibroin fabrics and the glass cover slip controls were all sterilized by exposure to 75% aqueous ethanol for 1 h and soaked with PBS solution for an additional 2h. The sterilized samples were then placed in triplicate in a 24-well tissue culture plate and incubated with 1 mL FBS (0.1%) solution (selected from absorbance curve, Figure S4, Supporting Information) for 24 h at 37°C. The concentrations of FBS were measured before and after adsorption using a Lambda 25 UV-visible spectrophotometer (Perkin Elmer, USA) at 280 nm with the aid of a standard FBS calibration curve (Figure S5, Supporting Information) that had been established previously. Platelet adhesion assay: Blood was drawn from a healthy volunteer and immediately mixed with acid citrate dextrose (ACD) solution. The anticoagulated whole blood was then centrifuged at 1500 rpm (revolution per minute) for 15 min to obtain platelet rich plasma (PRP). The untreated and modified silk fibroin fabrics were cut into small pieces (1×1 cm2), which were allowed to contact with 75µL of PRP for 3 h at 37°C under the static conditions. After washing with PBS, fabrics were fixed using 2% glutaraldehyde solution for 30 min; followed by washing with PBS and immersed into 55, 70, 80, 90, 95 and 100% ethanol solution in sequence and finally dried in a desiccator. Samples were then gold-sputtered for examination with SEM. Three samples each of the untreated and modified silk fibroin fabrics were examined, and the typical results obtained were presented.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 34

Statistical analysis The data are reported as means and standard deviations, and the error bars in the figures correspond to one standard deviation. All the statistical analyses were performed using a one way analysis of Variance (ANOVA) statistic. A p value of < 0.05 was selected as the confidence interval where differences were first found to be significant. The data in the tables are indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001. Results & discussion Surface morphology The surface of the modified silk fibroin fabrics (SFF-3 and SFF-5), as seen from the SEM micrographs converted from very smooth [Figure 3(a1, a2)] to uneven and bumpy [Figure 3(b1, b2, c1, c2)].The 3 layers of polyelectrolyte deposition and heparin immobilization raised miniature spherical form on the fibroin fabric surfaces. The 5 layers of polyelectrolyte deposited and heparin immobilization has rough surface, and also has raised globular appearance, but the size of each globule areas (convex protrusions) appears larger, and the distribution of raised globular areas is not uniform. The irregular distribution of separate “hills and valleys” suggest that the polyelectrolyte distribution was not applied evenly across the whole fiber surface. In some areas the discrete globular regions have combined and fused together to form a newly created and extensive modified surface. Figure 4 shows the AFM images of the unmodified and surface treated silk fibroin fabrics. The surface of the fibroin fabric seemed to change as a consequence of polyelectrolytes deposition and heparin immobilization. The surface of untreated silk fibroin fabric (SFF)(Figure 4a) appeared to be very smooth. However, the modified fabric surfaces (SFF-3 and SFF-5) (Figure


Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

4b, c) appeared to be more uneven with numerous curved bulges divided by “pits or valleys” after surface treatment, recommending that the application of polyelectrolytes resulted sporadic condensation bunches on the fiber surface. Identification of the presence of heparin on the modified silk fibroin fabrics surface Figure 5 presents the element analysis using EDX of the untreated (SFF) and surface modified silk fibroin fabrics (SFF-3 and SFF-5). The presence of both sulfur (S) and sodium (Na) peaks in EDX were identified in both SFF-3 and SFF-5 (Figure 5b, c) whereas, no such peaks are evident in SFF (Figure 5a). The presence of both S and Na peaks confirm that the heparin has successfully immobilized onto silk fibroin fabrics. The peaks owing to C and O atoms appeared in both untreated and modified fabrics approximately at 0.30 and 0.50 keV, respectively, as expected, but the signal due to Na atom approximately at 1 keV and S atoms at approx. 2.15 keV are observed for both of the modified samples. The S peak is linked to the existence of sulfated moieties. In this case, it was due to the EDC/NHS activated immobilized heparin on the polyelectrolytes deposited silk fibroin fabrics. These outcome are in accord with the formerly published research paper.30 Figure 5(d) shows the surface atomic relative ratios measured by EDX analysis in untreated and modified silk fibroin fabrics and heparin. The presence of heparin also identified on the SFF-3 and SFF-5 by measuring their absorbance in a UV-vis NIR spectrometer (U-4100, Hitachi, Japan). Alcian Blue shows two distinctive peaks at about 630 and 700 nm. SFF-3 and SFF-5 also show (Figure 6) also two peaks like the Alcian Blue. Whereas, SFF shows no evidence of such peaks. The presence of two peaks on the modified samples indicates that heparin was immobilized on the silk fibroin fabric surface and hence reacted with the Alcian Blue.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 34

Water contact angle results Figure 7(a) shows the results of the contact angle tests by the static captive bubble method. Significant difference (p < 0.001) in the contact angle values can be observed between SFF to SFF-3 and SFF to SFF-5. Significant difference (p < 0.01) in the contact angle value is also observed between SFF-3 to SFF-5. SFF shows an average contact angle of about 50°, whereas, it decreased to 38% and 44% for SFF-3 and SFF-5, respectively, indicating a highly hydrophilic surface. The water contact angle measured in this method also proves that deposition of 5 layers followed by heparin immobilization fabric retains higher hydrophilic property than 3 layers deposition. Unlike the captive bubble method, the sessile drop method is also a suitable and reliable technique to measure the contact angle of substrate. As surface wettability of the modified silk fibroin fabrics was significantly enhanced by surface treatment, the static contact angle measurements were very difficult to measure during measurement. All the modified silk fibroin fabrics showed a quicker absorbance time as the drop of water was absorbed without delay onto their surfaces. Figure 7(b) shows the dynamic contact angles results obtained from sessile drop method. It took about 250 ms for the water droplet to absorb completely onto the untreated control sample surface, however, the entire water droplets were completely absorbed into the surface of the modified fibroin fabric in 179.2 ms (SFF-3) and 99.5ms (SFF-5), respectively. A maximum water contact angle of 50.5° was recorded for the unmodified silk fibroin fabric in comparison with the lower values of 40.0° and 23.2° for the modified SFF-3 and SFF-5 surfaces respectively. It took about 69 ms for the SFF to arrive at a contact angle of 32.3°, while modified SFF-3 fabric


Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

shows water contact angle of 17.3° at this time. After that it was not possible to take the image of contact angle of SFF-3 due to the hydrophilicity. SFF-5 sample attributed a contact angle of 14.5° after 23 ms of the exposure of a drop of water onto its surface. Afterward unlike SFF-3, the water droplet was disappeared on its surface so quickly that other images of the contact angles were not possible to measure. The values of contact angle measurements from water and ethylene glycol were adopted to compute the surface free energy presented in Figure 7(c). Both surface modified samples demonstrated a tiny dissimilarity between the dispersion component, γd which is dependent directly on the number of the nonpolar functional groups. However a significant difference in polar component, γp, which depends on the number of the polar functional groups, was obtained between them. Both modified samples (SFF-3 and SFF-5) showed a significant increase of the polar component and a decrease of the disperse component of the surface free energy compared to untreated sample (SFF). An increase in surface free energy proves the presence of polar groups in the outer layer of the samples. As expected, the deposition of polyelectrolytes self-assembly could promote the addition of functional groups, such as COO- and NH3+ on the silk fibroin fabric surfaces and that could be the motive for the enhanced surface hydrophilicity of the surface treated samples. The hydrophilicity was additional improved by the application of EDC/NHS activated low molecular weight heparin, which being a sulfated glycosaminoglycan, contains abundant carboxyl (COO-) and sulfate (OSO3-) hydrophilic groups along its structure. Bursting strength Sufficient mechanical strength is critical for the long term stability of the small diameter vascular graft. Figure 8(a) shows the results of the bursting strength tests of untreated (SFF) and modified


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 34

silk fibroin fabrics (SFF-3 and SFF-5). No significant difference (p > 0.05) in the bursting strength can be found between the SFF and the experimental groups (SFF-3 and SFF-5) and also between SFF-3 and SFF-5. Compared to the SFF, SFF-3 and SFF-5 samples showed a negligible loss of bursting strength of about 1.12% and 1.41% upon modification for SFF-3 and SFF-5, respectively. The small amount of loss of bursting strength of the modified samples could be attributed due to two reasons. Firstly the polyelectrolytes was applied onto silk fibroin fabric in slightly acidic condition (PAH at pH 5 and PAA at pH 4). In such acidic modification condition silk fibroin fabric could lose some of its strength.31 Secondly, XRD results (Figure 8b) shows that due to the polyelectrolytes deposition and heparin immobilization, small amount of crystallinity % have been reduced. The crystallinity % of SFF, SFF-3 and SFF-5 was calculated as 39.16%. 37.27% and 36.04%, respectively. Heparin is a highly amorphous polymer (crystallinity % of 6.2) and hence could influence the crystallinity % of modified samples. So the changes of crystallinity could also be the reason of some strength loss of modified silk fibroin fabrics. Protein adsorption on the untreated and modified silk fibroin fabrics surface Significant difference (p < 0.001) of the amount of adsorbed fetal bovine serum (FBS) protein were obtained between the untreated (SFF) and experimental groups (SFF-3 and SFF-5) (Figure 9). However, no significant difference (p > 0.05) of protein adsorption was found between SFF-3 and SFF-5. The adsorbed amount of FBS was decreased by 55% (SFF-3) and 59% (SFF-5) correspondingly in comparison with SFF sample. The fewer amount of protein adsorption found on the modified fabric surfaces compared to the SFF surface, possibly due to electrostatic


Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

repulsion. The prevalence of sulfate and carboxylate groups endows heparin with a high negative charge (approximately -75), which mediates its electrostatic interactions with many proteins.32 Platelet adhesion test result The attraction of platelets to blood-contacting surface straightforwardly prejudiced the hemocompatibility of vascular prosthesis. Upon sticking platelets on a surface, they become activated and grouped together to form thrombus, and then slowly reduce blood flow, resulting blocking of the blood vessel.33 Scanning electron micrographs of unmodified (SFF) and surface treated silk fibroin fabrics (SFF-3 and SFF-5) (Figure 10) after being contacted with PRP is presented here, which clearly shows that the surface of the SFF is enclosed with an accumulation of platelets. Most of the platelets have undergone a morphological change from discs to spherical cells with pseudopodia and finally completely aggregated and activated on the untreated fabric surfaces [Figure 10(a1, a2)]. In contrast, very few platelets are shown on the modified silk fibroin fabrics surface [Figure 10(b1, b2, c1, c2)]. In the modified fabric surfaces, platelets are showed a round shape with no pseudopodia, which reflects the less activated state of the adhered platelets, as shown by SEM. This in vitro experiment clearly demonstrates that modified fabrics could restrain platelet adhesion and activation showing undesirable thrombus development. Two possible reasons are being considered for the reduced thrombogenicity of the surface modified silk fibroin fabrics. In the first place, the immobilized heparin diminishes the arrangement of thrombin in view of the hindrance of coagulation. Thus, both fibrin development and thrombin-interceded platelet enactment are reduced. Second, the adversely charged surface straightforwardly averts platelet connection with the modified silk fibroin fabrics due to


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

electrostatic repugnance, in light of the fact that the net charge of the platelet is likewise profoundly negative.34 In addition, since heparin is believed to be immobilized by electrostatic force, therefore it’s a question whether the electrolytes in blood could remove it from the surfaces of the materials or not. To address this issue, it can be anticipated that there is a negligible influence of the blood electrolyte on the immobilized heparin. In our previous study, we have carried out the rate of coagulation assay using untreated and surface modified silk fibroin fabrics. Strong electrolyte, calcium chloride (CaCl2) was used during the experiment to activate the blood coagulation process. Results revealed that all the heparin immobilized silk fibroin fabrics possessed slower rates of coagulation than the unmodified fibroin samples, suggesting the effectiveness of immobilized heparin against electrolyte.35 Conclusion EDC/NHS activated heparin was immobilized onto the polyelectrolytes deposited silk fibroin fabric to develop a functionalized silk fibroin surface that can elicit specific biological responses such to resist protein adsorption and platelets adhesion. The developed surface modification process was achieved by layer-by-layer deposition of 3 and 5 layers polyelectrolyte on the silk fibroin surfaces. SEM and AFM demonstrated that the utilization of 5 layers and then EDC/NHS activated heparin treatment resulted a rougher surface topography compared to the 3 layers technique. The results of this investigation suggest that the incorporation of heparin, onto silk fibroin surface would allow exploitation of heparin’s affinity in improved hydrophilicity and less protein adsorption. Thrombosis following implantation of small caliber synthetic vascular grafts is a major cause of graft failure. Surface modified silk fibroin fabrics showed strong resistance of


Page 16 of 34

Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

platelet adhesion indicating good antithrombosis property. Apart from the improved biological performance, the surface modification approach of silk fibroin fabric showed less influence on it bursting strength. EDX results ensured the presence of heparin onto the modified silk fibroin fabrics. Thus we conclude that the surface modification with the deposition of 5 layers of polyelectrolyte followed by heparin immobilization is more effective in contrast with the 3 layers and hence this modification technique could be used for the modification of small diameter silk fibroin based vascular prostheses. Acknowledgments We gratefully acknowledge the financial support by the National Natural Science Foundation of China (Grant No.31100682 and No. 81371648), the Fundamental Research Funds for the Central Universities and the 111 Project “Biomedical Textile Materials Science and Technology” (Grant No.B07024). Special thanks and appreciation are extended to Dr. Ying MA, Shen Gaotian, Dong Ting, Xu Lili, Wang Yiying, Wang Yexiang, Guan Ying, Li Chaojing, Fu Yijun regarding technical and experimental assistance. Supporting information available: Additional information regarding chemical structures of the used materials, contact angle instrument setup by captive bubble and sessile drop methods, absorbance of FBS protein solution at different concentration, calibration curve of FBS protein and calibration curve of heparin. This information is available free of charge via the Internet at http://pubs.acs.org/.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 34

References (1)

Montdargent, B.; Letourneur, D. Toward new biomaterials. Infect. Control Hosp.

Epidemiol.2000, 21, 404-410. (2)

Roach, P.; Farrar, D.; Perry, C. C. Interpretation of protein adsorption: surface-

induced conformational changes. J. Am. Chem. Soc.2005, 127, 8168-8173. (3)

Liu, J.; Wang, Q.; Fan, X. R.; Sun, X. J.; Huang, P. H. Layer-by-Layer Self-

Assembly Immobilization of Catalases on Wool Fabrics. Appl. Biochem. Biotechnol.2013, 169, 2212-2222. (4)

Cheng, C.; Ma, L.; Wu, D.; Ren, J.; Zhao, W.; Xue, J.; Sun, S.; Zhao, C.

Remarkable pH-sensitivity and anti-fouling property of terpolymer blended polyethersulfone hollow fiber membranes. J. Membr. Sci.2011, 378, 369-381. (5)

Tan, D.; Zhang, X.; Li, J.; Tan, H.; Fu, Q. Modification of poly(ether urethane)

with fluorinated phosphorylcholine polyurethane for improvement of the blood compatibility. J. Biomed. Mater. Res., Part A2012, 100 A, 380-387. (6)

Steen, M. L.; Jordan, A. C.; Fisher, E. R. Hydrophilic modification of polymeric

membranes by low temperature H2O plasma treatment. J. Membr. Sci.2002, 204, 341-357. (7)

Theapsak, S.; Watthanaphanit, A.; Rujiravanit, R. Preparation of chitosan-coated

polyethylene packaging films by DBD plasma treatment. ACS Appl. Mater. Interfaces2012, 4, 2474-2482. (8)

Faber, H.; Hirschmann, J.; Klaumünzer, M.; Braunschweig, B.; Peukert, W.;

Halik, M. Impact of oxygen plasma treatment on the device performance of zinc oxide nanoparticle-based thin-film transistors. ACS Appl. Mater. Interfaces2012, 4, 1693-1696.


Page 19 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(9)

Chang, Y.; Shih, Y. J.; Ruaan, R. C.; Higuchi, A.; Chen, W. Y.; Lai, J. Y.

Preparation of poly(vinylidene fluoride) microfiltration membrane with uniform surfacecopolymerized poly(ethylene glycol) methacrylate and improvement of blood compatibility. J. Membr. Sci.2008, 309, 165-174. (10)

Huang, X. J.; Guduru, D.; Xu, Z. K.; Vienken, J.; Groth, T. Blood Compatibility

and Permeability of Heparin-Modified Polysulfone as Potential Membrane for Simultaneous Hemodialysis and LDL Removal. Macromol. Biosci.2011, 11, 131-140. (11)

Fink, H.; Hong, J.; Drotz, K.; Risberg, B.; Sanchez, J.; Sellborn, A. An in vitro

study of blood compatibility of vascular grafts made of bacterial cellulose in comparison with conventionally-used graft materials. J. Biomed. Mater. Res., Part A2011, 97 A, 52-58. (12)

Yi, Z.; Zhu, L.; Cheng, L.; Zhu, B.; Xu, Y. A readily modified polyethersulfone

with amino-substituted groups: Its amphiphilic copolymer synthesis and membrane application. Polymer2012, 53, 350-358. (13)

Yi, Z.; Zhu, L. P.; Xu, Y. Y.; Zhao, Y. F.; Ma, X. T.; Zhu, B. K. Polysulfone-

based amphiphilic polymer for hydrophilicity and fouling-resistant modification of polyethersulfone membranes. J. Membr. Sci.2010, 365, 25-33. (14)

Jiang, J. H.; Zhu, L. P.; Li, X. L.; Xu, Y. Y.; Zhu, B. K. Surface modification of

PE porous membranes based on the strong adhesion of polydopamine and covalent immobilization of heparin. J. Membr. Sci.2010, 364, 194-202. (15)

Sperling, C.; Houska, M.; Brynda, E.; Streller, U.; Werner, C. In vitro

hemocompatibility of albumin-heparin multilayer coatings on polyethersulfone prepared by the layer-by-layer technique. J. Biomed. Mater. Res., Part A2006, 76, 681-689.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(16)

Page 20 of 34

Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Biomedical applications of layer-

by-layer assembly: From biomimetics to tissue engineering. Adv. Mater.2006, 18, 3203-3224. (17)

Srivastava, S.; Kotov, N. A. Composite Layer-by-Layer (LBL) assembly with

inorganic nanoparticles and nanowires. Acc. Chem. Res.2008, 41, 1831-1841. (18)

Zhang, H.; Shih, J.; Zhu, J.; Kotov, N. A. Layered nanocomposites from gold

nanoparticles for neural prosthetic devices. Nano Lett.2012, 12, 3391-3398. (19)

Kim, Y.; Zhu, J.; Yeom, B.; Di Prima, M.; Su, X.; Kim, J. G.; Yoo, S. J.; Uher, C.;

Kotov, N. A. Stretchable nanoparticle conductors with self-organized conductive pathways. Nature2013, 500, 59-63. (20)

Yan, S.; Zhang, K.; Liu, Z.; Zhang, X.; Gan, L.; Cao, B.; Chen, X.; Cui, L.; Yin, J.

Fabrication of poly(L-glutamic acid)/chitosan polyelectrolyte complex porous scaffolds for tissue engineering. J. Mater. Chem. B2013, 1, 1541-1551. (21)

Baguneid, M. S.; Seifalian, A. M.; Salacinski, H. J.; Murray, D.; Hamilton, G.;

Walker, M. G. Tissue engineering of blood vessels. Br. J. Surg.2006, 93, 282-290. (22)

Lovett, M.; Cannizzaro, C.; Daheron, L.; Messmer, B.; Vunjak-Novakovic, G.;

Kaplan, D. L. Silk fibroin microtubes for blood vessel engineering. Biomaterials2007, 28, 52715279. (23)

Kannan, R. Y.; Salacinski, H. J.; Edirisinghe, M. J.; Hamilton, G.; Seifalian, A. M.

Polyhedral oligomeric silsequioxane-polyurethane nanocomposite microvessels for an artificial capillary bed. Biomaterials2006, 27, 4618-4626. (24)

Nakazawa, Y.; Sato, M.; Takahashi, R.; Aytemiz, D.; Takabayashi, C.; Tamura,

T.; Enomoto, S.; Sata, M.; Asakura, T. Development of small-diameter vascular grafts based on


Page 21 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

silk fibroin fibers from bombyx mori for vascular regeneration. J. Biomater. Sci. Polym. Ed.2011, 22, 195-206. (25)

Yang, X.; Wang, L.; Guan, G.; King, M. W.; Li, Y.; Peng, L.; Guan, Y.; Hu, X.

Preparation and evaluation of bicomponent and homogeneous polyester silk small diameter arterial prostheses. J. Biomater. Appl.2014, 28, 676-687. (26)

Enomoto, S.; Sumi, M.; Kajimoto, K.; Nakazawa, Y.; Takahashi, R.; Takabayashi,

C.; Asakura, T.; Sata, M. Long-term patency of small-diameter vascular graft made from fibroin, a silk-based biodegradable material. J. Vasc. Surg.2010, 51, 155-164. (27)

Elahi, M. F.; Guan, G.; Wang, L.; King, M. Influence of Layer-by-Layer

Polyelectrolyte Deposition and EDC/NHS Activated Heparin Immobilization onto Silk Fibroin Fabric. Materials2014, 7, 2956-2977. (28)

Podsiadlo, P.; Qin, M.; Cuddihy, M.; Zhu, J.; Critchley, K.; Kheng, E.; Kaushik,

A. K.; Qi, Y.; Kim, H.-S.; Noh, S.-T.; Arruda, E. M.; Waas, A. M.; Kotov, N. A. Highly Ductile Multilayered Films by Layer-by-Layer Assembly of Oppositely Charged Polyurethanes for Biomedical Applications†. Langmuir2009, 25, 14093-14099. (29)

Rudawska, A.; Jacniacka, E. Analysis for determining surface free energy

uncertainty by the Owen–Wendt method. Int. J. Adhes. Adhes.2009, 29, 451-457. (30)

Cestari, M.; Muller, V.; Rodrigues, J. H. d. S.; Nakamura, C. V.; Rubira, A. F.;

Muniz, E. C. Preparing Silk Fibroin Nanofibers through Electrospinning: Further Heparin Immobilization toward Hemocompatibility Improvement. Biomacromolecules2014. (31)

Sheikh, M. R.; Farouqui, A. N.; Yahya, R.; Hassan, A. Effect of acid modification

on dyeing properties of Rajshahi silk fabric with different dye classes. Fibers Polym.2011, 12, 642-647.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32)

Page 22 of 34

Capila, I.; Linhardt, R. J. Heparin - Protein interactions. Angew. Chem., Int.

Ed.2002, 41, 390-412. (33)

Okada, T.; Ikada, Y. Modification of silicone surface by graft polymerization of

acrylamide with corona discharge. Die Makromolekulare Chemie1991, 192, 1705 - 1713. (34)

Han, F.; Dong, Y.; Su, Z.; Yin, R.; Song, A.; Li, S. Preparation, characteristics

and assessment of a novel gelatin–chitosan sponge scaffold as skin tissue engineering material. Int. J. Pharm.2014, 476, 124-133. (35)

Elahi, M. F.; Guan, G.; Wang, L.; King, M. W. Improved hemocompatibility of

silk fibroin fabric using layer-by-layer polyelectrolyte deposition and heparin immobilization. J. Appl. Polym. Sci.2014, 131, 9307-9318.


Page 23 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure captions

Figure 1. Illustration of the Initiation of coagulation cascade and fibrin clot formation. Figure 2. Schematic illustration of the silk fibroin fabric modification process. Figure 3. SEM micrographs of untreated and surface modified silk fibroin fabrics: (a1, a2) SFF, (b1, b2) SFF-3 and (c1, c2) SFF-5. Magnification: top row ×5,000; bottom row ×10,000. Figure 4. AFM images of untreated and surface modified silk fibroin fabrics: (a) SFF; (b) SFF-3 and (c) SFF-5. Figure 5. EDX spectra of untreated and surface modified silk fibroin fabrics: (a) SFF; (b) SFF-3, and (c) SFF-5. Figure 6. UV-vis NIR spectra of Alcian Blue solution, and Alcian Blue stained untreated and surface modified silk fibroin fabrics. Figure 7. Contact angles measured by (a) captive bubble and (b) sessile drop methods. Data for each sample are expressed as the mean (n = 5). The error bar = 1standard deviation. Statistical differences indicated with (**) for p< 0.01, and (***) for p< 0.001. Figure 8. (a) Bursting strength and (b) XRD patterns of the untreated and surface modified silk fibroin fabrics. Data of bursting strength for each sample are expressed as the mean (n = 5). The error bar = 1standard deviation. Figure 9. Average amount of FBS adsorbed (mg/ mg substrate) on the untreated and surface modified silk fibroin fabrics. Statistical differences between the untreated (SFF) and the modified silk fibroin fabrics (SFF-3, SFF-5) are indicated with (***) for p < 0.001. Data for each sample are expressed as the mean (n = 3). The error bar = 1standard deviation.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10. SEM micrographs of platelet adhered untreated and surface modified silk fibroin fabrics: (a1, a2) SFF, (b1, b2) SFF-3 and (c1, c2) SFF-5. Magnification: top row ×1, 000; bottom row ×5, 000.


Page 24 of 34

Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2


Page 26 of 34

Page 27 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4


Page 28 of 34

Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6


Page 30 of 34

Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 7


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8


Page 32 of 34

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 9


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10


Page 34 of 34

Surface Modification of Silk Fibroin Fabric Using Layer-by-Layer

Recommend Documents