Development and Processing of Novel Heparin Binding Functionalized Modified Spider Silk Coating for Catheter Providing Dual Antimicrobial and Anticoagulant Properties

Abstract

Tailored surface coatings have been used for decades to improve material performance in blood. Among different approaches, heparin based biomedical coatings have found great success in the commercial catheter market. However, they have their own limitations. Coating of a vascular device with a heparin binding peptide (HBP), which can sequester the circulating heparin, presents numerous advantages over both systemic heparin therapy and direct heparin bound surfaces. Embedding HBP in a silk biopolymer provides the mechanical integrity necessary under dynamic flow conditions to both insert the catheter and maintain proper blood flow. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property, a critical and unique aspect to combat catheter related blood stream infections and extend the longevity of hemodialysis catheters. To assess this hypothesis, a recombinant fusion protein (S₄H₄) containing both silk amino acid motifs and HBP was assessed as a coating on a silicone surface. After validating that, the protein was deposited on the surface via XPS, Raman spectroscopy, ATR and SEM imaging, antimicrobial and anticoagulant activities were evaluated. The coating was able to prevent not only planktonic bacterial growth but also prevented the growth of a biofilm. Finally, the coating had both antibacterial and anticoagulant effect simultaneously. This study proves the successful production of a silk-based biopolymer that can be embedded with a heparin-binding functionality to create a dual functional device coating that can prevent infection and thrombosis together.

Graphical Abstract

Spider silk is functionalized with heparin binding peptide (HBP) and coated on catheter tubing which can capture endogenous heparin to make it anti-infective and anti-thrombotic surface

Introduction

Thrombosis (formation of a blood clot) and infection are frequently inextricably connected, effectively countering resolution with single pathology therapeutics, allowing both pathologies to proceed unabated [1]–[3]. Indeed, hemostatic abnormalities are encountered in most cases of infection, ranging from an increase in sensitive markers for active coagulation to a rise in host factors such as platelets, fibrin and fibronectin that may result in localized thrombotic complications; simultaneously, independent production of thrombi can also provide a rich matrix of fibrils for bacterial adhesion [4], [5]. This mechanism is of particular importance in cases of CRBSI (catheter-related bloodstream infection) where thrombi lead to microbial surface colonization of catheters or medical devices [6]. Importantly, when bacteria colonize a surface, such as an in dwelling biomedical device, they often secrete a complex mucopolysaccharide barrier to form a “biofilm”, allowing them to not only escape the host immune response but also to thwart many pharmaceutical interventions and enhance their virulence [7], [8].

There are 5 main clinical interventions for thrombosis- related flow problems: forceful catheter flush, fibrinolytic enzymes, mechanical thrombus removal, catheter exchange, and percutaneous fibrin-sheath stripping [9], [10]. Systemic anticoagulation therapy and heparin-based biomedical coatings have been used for decades to improve material performance in the blood and biological milieu; however, heparin therapy can simultaneously promote and treat the thrombosis [11], [12]. Systemic therapy may cause Heparin-induced thrombocytopenia (HIT) as a potential complication that can lead to a non-immune mediated transient decrease in platelet count but a rise in levels of fibrinogen. In addition, studies evaluating catheter surface treatments to reduce infection have provided inconsistent results in reducing CRBSIs. Traditionally, either systemic antibiotic administration with antibiotic lock therapy or heparin therapy has been employed separately for the treatment of CRBSI. The major drawback of the antibiotic lock therapy is the development of antibiotic resistance by the bacteria in the biofilm due to sub therapeutic exposure of bacteria to the antibiotics while only eliminating the intraluminal source of infection [13], [14]. Furthermore, such a strategy addresses only one problem at a time and completely ignoring the complex relationship between infection and thrombosis, either of which can limit the number of in dwelling catheter days.

Alternatively, recent advances in catheter materials and the relative success of chemically defined polymer surface coatings have proved promising carriers for pharmaceutical immobilization to treat CRBSI. Surface modification and coating of the catheter material can be done via either a passive approach by changing the surface chemistry or physical structure of the device or by a bioactive approach by locally releasing a pharmacological agent to inhibit coagulation. Heparin has been employed in various forms as a covalently immobilized surface coating or a prophylactic agent to improve hemocompatibility of medical devices [15], [16]. Unfortunately, the lifetime of these surface modifications can be limited by the natural foreign body response, which can lead to fibrous capsule formation. This necessitates a resilient and durable strategy to provide improved device resistance to biofouling and device longevity at the same time. A “self-renewable” multi-functional coating relying on biological mechanisms and the coagulation-signaling cascade may prove an effective, alternative strategy.

In addition to biological longevity, mechanical integrity under dynamic flow conditions is a key factor for coating materials. Although many current catheter bulk materials possess sufficient mechanical function, the coatings lack the strength and flexibility necessary to resist delamination during insertion of the catheter while the bulk material maintains proper blood flow. In this context, genetically modified silk protein, which can capture endogenous heparin tethering it to the device surface, provides an attractive opportunity as a biomaterial coating.

Silk fibroin has demonstrated a combination of excellent properties including biocompatibility, robust mechanical strength in various material formats and controllable rates of degradation to non-toxic byproducts in vivo, leading it to be increasingly considered for various biomedical and drug delivery applications [17], [18]. Additionally, the ability to manipulate the primary structure/function relationship of spider silk by embedding different functional amino acid blocks or by altering the ratio of amino acid blocks [19], [20] to produce a mechanically robust and chemically defined biomaterial makes synthetic spider silk proteins an attractive biomaterial. One such attractive target amino acid block to endow new biological function to a recombinant silk peptide is found in the heparin binding region of a variety of endogenous biomolecules (e.g., growth factors). Homology modeling of heparin binding regions reveals a structural prerequisite and consensus amino acid sequence, XBBBXXBX, where B is a basic residue (arginine) and X represents a hydrophobic or uncharged amino acid (alanine) [21], [22]. Interestingly, this consensus sequence has several tertiary structures analogous to antimicrobial peptides and is thought to also be antibacterial [21], [23], [24]. The heparin binding peptide (HBP) (ARKKAAKA) can be either covalently attached to the silk peptide or genetically linked to the silk sequence. This fusion protein is proposed to mitigate both device-centered thrombosis and bacterial biofouling using naturally occurring immobilized amino acid motifs. In this paper, we report the use of this fusion protein as a silicone material coating to prevent both coagulation and bacterial adhesion.

MATERIALS AND METHODS:

Protein synthesis:

The genetic sequence of the fusion protein containing four repeats of a general MaSp2 like monomer (GPGXXA₈ where X = G, Q, Y [25]) and four repeats of heparin binding (ARKKAAKA) motif (S₄H₄) was synthesized and cloned into pET 30a (Genscript, Piscataway, NJ). The recombinant protein was expressed in E. coli (New England Biolabs) and purified by nickel affinity chromatography [26]. This recombinantly expressed protein was validated by dot blot against anti-His antibody and its molecular weight was confirmed by SDS-PAGE and mass spectrometry (data not shown). The purified protein was dialyzed for further purification and lyophilized.

Coating preparation:

Silicone sheets (USP class VI Silicone, thickness − 0.01”, Specialty Manufacturing inc, Saginaw, MI) were used as to mimic the bulk catheter material. The silicone sheet was cut into square shaped coupons (25mm × 25mm) and subsequently activated by plasma treatment prior to coating. The plasma etching process was performed on Trion Phantom RIE Plasma Etcher (Trion Technology, Clearwater, FL) at 50mTorr with 20 sccm oxygen flow for 90 seconds [27]. The activated silicone coupons were immediately dip coated in the S₄H₄ protein solution (in PBS at 300μM) manually for about ten times with intermittent air drying for 5 minutes in between dipping.

Coating Characterization

Contact angle:

Static contact angles of coated and uncoated silicone sheets were measured using FTÅ 125 Contact Angle/Surface Tension Analyzer (First Ten Angstroms, Portsmouth VA) to determine the wettability of the surface. Coated surfaces were dried prior to testing. Deionized water (10ul) was dispensed onto the surface capturing the image of the water drop using a charged coupled device (CCD) camera. Images were analyzed using an Fta32 V2.0 image analysis software.

X-ray photoelectron spectroscopy (XPS):

A K-Alpha™ X-ray Photoelectron Spectrometer (Thermo Scientific, MA) was used for elemental analysis to determine the surface chemistry of both coated and uncoated silicone surfaces. Survey spectra were obtained from −10 to 1350 eV with a pass energy of 200 eV and 1 eV energy step size. High resolution (HR) analyses with pass energy of 40 eV were performed at a take-off angle of 90° to determine elemental atomic percent composition. HR scans were performed at 50 eV pass energy for Oxygen (O 1s), Carbon (C 1s), and Nitrogen (N 1s) scans.

Scanning Electron Microscopy (SEM) imaging:

The thickness of the coating was analyzed using SEM. Coated and uncoated sheets were cut using a razor blade and the samples (n=3) were attached to cylindrical aluminum mounts using colloidal silver paint (Structure Probe Inc., West Chester, Pennsylvania). Subsequently, the samples were coated with a conductive layer of carbon in a high-vacuum evaporative coater (Cressington 208c, Ted Pella Inc., Redding, California). Images were obtained with a JEOL JSM-7600F scanning electron microscope (JEOL USA Inc., Peabody, Massachusetts) operating at 2 kV.

Attenuated total reflection (ATR):

ATR spectra of the coated and uncoated silicone tube were obtained with a Nicolet 6700 FTIR-ATR Spectrometer operated with OMNIC software [28]. The crystal used was a polished diamond. The silicone sheets were placed on the diamond crystal and the spectra were scanned from 400 to 4000 cm⁻¹. Spectra were recorded with 64 scans and 4 cm⁻¹ resolution with data spacing of 0.964 cm⁻¹. All spectra were reported without smoothing the output.

Raman Spectroscopy:

Confocal Raman spectra were obtained from Horiba Jobin Yvon’s Raman Spectrometer on LabRAM Aramis software equipped with He-Ne laser source emitting at 532nm [29]. The sample was scanned at a constant speed with 2μm step size. The radiation was focused in the Raman-shift range of 3500–300 cm⁻¹ and scattered radiation was collected with a confocal aperture of 600μm and a slit aperture of 150μm.

Characterization of antimicrobial properties

ATP assay:

An overnight S. aureus (ATCC 49230) culture was prepared in LB broth (Fischer Scientific, Hampton, NH). To create a biofilm, the overnight bacterial culture was taken and diluted into LB broth to get an OD₆₀₀=0.5. Later, S₄H₄ coated and non-coated silicone sheets (40.92 mm² area) were placed in the wells of a 24-well plate and 1.5 mL of diluted bacterial culture was added to the well. The plates were incubated for 24 hours at 37 °C with shaking. After incubation, the silicone coupons were collected and washed in 1X Phosphate Buffered Saline PBS, three times. The silicone coupons were wiped with a sterile cotton swab, which was subsequently immersed in PBS and vortexed to make a bacterial suspension. The ATP assay was performed using the BacTiter Glo kit (Promega, WI, USA) according to the manufacturer’s protocol. Briefly, the bacterial suspension in PBS was equilibrated to room temperature (25 °C) and 100 μL of the bacterial suspension was put in an opaque 96 well plate. BacTiter Glo reagent (100 μL) was added and mixed followed by incubation at 25 °C for 5 minutes. After the incubation, luminescence was measured using SpectraMax5 spectrometer at a wavelength of 560nm.

Colony assay:

Antibacterial activity was evaluated by colony assay. Bacterial biofilm was allowed to develop on the coated and uncoated silicone tubing (ID 3mm × OD 4mm, length 3cm, GoFlow systems, Amazon, Seattle, WA) in a method analogous to that for the silicone coupons as described above. After 24-hour incubation, the tubing was rinsed gently to remove any unadhered bacteria. The tubing samples were then vortexed and serial dilutions of the vortexed solution were plated on an agar and incubated overnight at 37 °C. The number of colonies were counted the next day.

Simultaneous evaluation of antimicrobial anti-coagulant properties:

An overnight culture of S. aureus was prepared in LB broth. To create a biofilm, OD₆₀₀ of 0.5 bacterial culture was prepared from the overnight culture. Protein coated and non-coated silicone sheets (40.92 mm² area) were placed in a 24 well plate and 1.5 mL of bacterial culture was added to the well. The plates were incubated for 24 hours at 37 °C in an incubator. After incubation, the bacterial suspension was removed and the edges of the sheet were wiped with a sterile cotton swab to remove any unadhered bacteria. Subsequently, the sheets were rinsed gently with PBS placed in a 24 well plate, and 200 μL of heparin (100 μg/mL in TBS) was added and incubated for 2 hours. Heparin was removed from the wells and the sheets were again gently rinsed with PBS. Subsequently, the bacterial and heparin exposed sheets were used for the aPTT (APTT XL Pacific Homeostasis Assay, Thermo Fischer, USA) assay to check the clot formation. Briefly, 0.1 mL of plasma was added initially to the sheets followed by 0.1 mL of APTT-XL reagent and the reaction was incubated for 5 min. Calcium chloride solution (0.1 mL of 0.02 M) was then added to initiate clot-like formation and clot-like formation was assessed by measuring OD₆₀₀ to determine the change in optical density of the solution.

Evaluation of coating under shear stress:

An in vitro flow system has been designed to simulate the in vivo conditions of a hemodialysis catheter under blood flow conditions. The device was created to resemble a cone and plate rheometer, with an ability to run for extended periods of time. The cone-shaped attachment causes the working fluid to apply shear stress to the materials being tested. The speed was set to 996 rpm (rotations per minute) creating a shear stress of 2.18 Pa, which is equivalent to the shear stress in a catheter with a radius of 1.6 mm and a flow rate of 350 mL/min. The cylinder is filled with plasma and the system was operated for 24 hrs. After 24 hrs the coated sheet was taken and assessed for simultaneous bacterial growth and clot formation as described above.

Statistical analysis:

SPSS 23 was used to perform statistical analysis. Student t-test was used to determine the significance difference between coated and uncoated samples. Statistical significance was defined by p<0.01 for colony assay and ATP assay, and p<0.001 for simultaneous evaluation of anticoagulant and antimicrobial properties.

RESULTS:

Validation of protein:

Theoretical mass of S₄H₄ was predicted to be 18538 Da which was further confirmed by mass spectrometry (data not shown). Recombinant protein was validated by dot blot (Fig 3) using anti-His antibody which recognizes the Histidine tag attached to the N-terminus. This confirms the recombinant expression and purification of the S₄H₄ fusion protein.

Figure 3:

Dot blot showing recombinant His tag Silk₂ was predicted to be 14,766 Da while His tag S₄H₄ was predicted to be 16,355 Da.

Characterization of coating:

Once the protein was synthesized and validated, it was solubilized in PBS at a concentration of 300 μM. This protein solution was used to coat the silicone sheets by dip coating.

Contact angle:

To determine the presence of the S4H4 coating on the surface of hydrophobic silicone, the surface wettability of the coated silicone coupon, the static water contact angle of the coated and uncoated samples was measured. The contact angle of the uncoated silicone sheet was found to be 103.35° ±2.3° while the protein coated sheets showed a reduced contact angle of 19.6° ±3.4 (Fig 4), indicating a significant shift from hydrophobicity to hydrophilicity. Note that the protein coating was dried on the silicone coupon prior to testing.

Figure 4:

contact angle measurement of uncoated sheet (left), and coated sheet (right)

X-ray photoelectron spectroscopy (XPS) measurements:

A shift in the hydrophobic nature of the silicone sheet indicates a change in the surface, likely attributed to the presence of the protein; however, increased specificity was obtained by analyzing the chemical composition of the surface using XPS spectra. Both coated and uncoated silicone sheets show similar percentage composition for C1s, O1s while percentage of N1s increased from 1.56% to 6.1%. Importantly the percentage of silicone also decreased from 2.26% to 0.91% after coating (Table 1), further suggesting the presence of protein on the coated sheet.

Table 1:

XPS survey of the coated and uncoated silicone sheets with atomic percentages

Name	Atomic %
	Coated	Uncoated
C1	71.25	77.75
O1	16.58	14.86
N1	6.12	1.57
Cl2	3.4	3.18
Si2	0.91	2.26

Raman spectroscopy:

A shift in the chemical composition of silicone sheet surface was evidence of protein coating; however, the coating of tube structure similar to a catheter was assessed by comparing the Raman spectra of both uncoated and the coated tubing. In comparison to the uncoated tubing, Raman spectra of the coated tubing resulted in additional prominent peaks corresponding to Raman shifts at 788, 857, 1261, 1406, and 2795 cm-1 (Fig 5). The presence of doublet peaks at 788, 857 cm-1 confirms the presence of a protein with tyrosine as a key aromatic amino acid in the sequence. Similarly, the peak at 1261 cm-1 resulted from the amide linkage that is part of the α-helix structure of protein macromolecule confirming that coating has been deposited on the silicone sheet.

Figure 5:

Confocal Raman spectra of the coated silicone sheet showing the Raman shifts for tyrosine and amide bond compared to the uncoated silicone sheet

ATR:

Presence of the coating on the silicone tube can be augmented for the data obtained from ATR. As compared to the non-coated silicone tube, coated silicone tube resulted detectable peaks at wavenumbers 3268 and 1724 cm⁻¹ corresponding to the -OH (stretch), C=O (Stretch) (Fig 6) of tyrosine and glutamine. Reflectance peaks at 1632, and 1532 cm⁻¹ confirms the presence of aromatic C=C bond. Overall, ATR data further concludes that the coating was deposited on the silicone surface.

Figure 6:

ATR spectra of the coated silicone sheet compared to the uncoated sheet showing the -OH (stretch), C=O (Stretch) and aromatic C=C bond

SEM imaging:

Finally, the uniformity in coating and the thickness of the coating was examined by SEM imaging (Fig 7). Based on the imaging, the protein was found to be non-uniformly distributed, although it was clearly present. The thickness was found to be 10.2±3 μm as measured from the surface of the sheet.

Figure 7:

SEM imaging on the (A) coated tube (B) non coated tubes (C) thickness of the coating

Characterization of Bioactivity of the Protein Coating

Colony assay:

Initially, the antimicrobial activity of the coating was analyzed by counting the number of colonies adhered to the surface after incubation with a planktonic bacterial culture. The number of colonies on the protein-coated, silicone sheets was 5 ×10⁴ CFU/ml whereas the uncoated samples showed 3 × 10⁶ CFU/ml (n = 5) (Fig 8 LHS). The coated samples showed significantly lower number of colonies (p<0.01) when compared to the uncoated samples.

Figure 8:

Colony assay on the LHS and ATP assay on the RHS to show antibacterial activity of the coating

ATP assay:

After establishing the antibacterial activity of the coating against planktonic bacteria, the ability of the coating to prevent a biofilm was also evaluated using an ATP assay. Luminesce (RLU) was calculated per unit area (mm²) of the silicone sheets and was significantly lower (p<0.01, n=4) for S₄H₄ coated sheets compared to the non-coated samples (Fig 8 RHS).

Simultaneous evaluation of antimicrobial anti-coagulant properties:

After establishing that the coating had both antimicrobial and antithrombotic activity, it was evaluated to see if both activities could be achieved simultaneously. Initially, a biofilm was grown on both the protein-coated surface and an uncoated control. After rinsing the surface, an aPTT assay was performed to assess if a clot could still be formed. The optical density of the protein coated samples at wavelength of 600 nm (n=3, p<0.001) (Figure 9) was found to be less than the uncoated samples. Similar assessment was performed after undergoing shear stress in an in vitro set up. Similar trend in decrease in optical density of the uncoated samples seen after shear stress which only proves that the coating is stable and intact even after exposure to shear stress that can be seen under blood flow conditions.

Figure 9:

(A) Simultaneous evaluation of antimicrobial and anti-coagulant properties of the coating before shear stress (B) Simultaneous evaluation of antimicrobial and anti-coagulant properties of the coating after shear stress

Discussion

The common complications of long-term vascular access catheters are 1) infection, 2) thrombus formation, and 3) mechanical failure leading to poor blood flow, making these key design parameters for any catheter modification. Among these complications, the incidence of catheter infections has been cited to be as high as 29.6% and while approximately 70% of deep vein thrombotic events of upper extremity are due to intravenous catheters [9], [30]. Unfortunately, clinical treatment regimens often only address the single pathology, although it is clear that infection and thrombosis often occur almost simultaneously. Nevertheless, several strategies focused on modifying the biology/material interface through bulk material improvements, surface modifications, or pharmaceutical additions have been exploited with increasing importance to combat either thrombosis or biofilm formation. Specifically, heparin immobilization by covalent bonding or ionic interaction has demonstrated success as a coating for a variety of biomedical devices. Regrettably, the unavailability of heparin’s active site due to its involvement in a covalent bond or heparin disassociating from an ionic linkage [31] have prevented the widespread success of these strategies. Finally, mechanical compliance of the catheter, analogous to the third arm of Virchow’s triad is fundamental to the longevity of vascular access catheters, and it cannot be compromised by the addition of any coating, regardless of its biological activity. To meet these design criteria, a recombinant fusion protein consisting of a spider silk amino acid motif and a heparin binding motif (S₄H₄) serves as a promising alternative, avoiding not only the drawbacks of heparin therapy but also providing the advantages of heparin (via likely electrostatic and ionic interactions) while concurrently delivering a balance of strength and flexibility from the silk backbone. The recombinant fusion protein was successfully cloned and expressed in E. coli. After purification with Ni affinity chromatography, the fusion protein was validated by dot blot using anti-His antibody and mass spectrometry. Both the experiments confirmed successful production of recombinant protein.

Silicone is the major choice of material for vascular access catheters [32]. However, the surface of silicone is not only inert but also hydrophobic [28], making it relatively unavailable for interaction with a coating material. Therefore, plasma oxygen treatment was employed to introduce reactive hydroxy groups onto the surface of the silicone [33], [34], as verified by increase in hydrophilicity of the silicone surface (data not shown). Plasma etching the surface of the silicone allowed the protein to bind to the surface, creating a coating. Contact angle measurements showed a substantial decrease in the contact angle after coating suggesting that silicone surface had become hydrophilic (Figure 4) [33]. This also indicated that both oxygen treatment and coating deposition contributed to the surface wettability of the silicone surface. Subsequently, the chemical structure and thickness of the coating was evaluated. X-ray electron spectroscopy was able to confirm the presence of a protein coating by determining the chemical composition of the surface of the coated silicone sheet (Table 1). Nitrogen content increased from 1 to 6% after coating, which is probably due to the amine groups from the side chains of lysine and arginine in heparin binding region of the protein coating. In addition, the silicone elemental composition percentage decreased, further confirming that the surface is covered by the protein. Raman spectroscopy was employed to determine the fingerprint regions of specific amino acids particularly symmetrical compounds. Of particular importance among the Raman spectral shifts is the region at 1260 cm⁻¹ (Figure 5), which shows the amide linkage and also the α-helix. The presence of this band shows that the secondary structure of the protein is not altered with the addition of HBM motifs in the spider silk backbone. Fingerprint regions of the protein were further confirmed by ATR spectroscopy (Figure 6) which showed C=O and aromatic C=C stretching of glutamine and tyrosine respectively. Next, SEM imaging was performed to evaluate the thickness of the coating. A cross section of the coated and non-coated silicone sheets was evaluated by SEM and the thickness was found to be 10μm (Figure 7). Coating prior to the plasma activation resulted in a thickness of nano scale (data not shown); whereas, plasma activation increased the thickness of coating further providing an advantage of thick coating.

The structural prerequisite of HBM is the presence of a basic residue separated by a consensus of hydrophobic residues [22], [35]. Segregation of discrete hydrophobic and cationic amino acid sectors in this consensus sequence is analogous to many identified antimicrobial peptides (AMPs) and thought to be fundamental to their bactericidal function allowing permeation of the bacterial membrane [21]. Most AMPs are broad spectrum and do not have a particular bacterial target, which minimizes the development of bacterial resistance to the coating. The activity of the fusion protein as an antibacterial coating material was evaluated by counting the number of colonies after incubation with bacteria. From figure 8, in the colony count assay, it is evident that in the presence of coating, the number of bacterial colonies was reduced significantly compared to the uncoated sheets. In addition to antimicrobial activity, ATP assay was performed to evaluate the bacterial cell viability (Figure 8). In this assay lower luminescence values corresponds to lower number of cells adhered to the surface. From the bar graph in figure 8, the coated sheets have a significantly lower number of adhered cells compared to the uncoated samples. This further confirms that the coating not only shows antibacterial activity but can also prevent the formation of biofilm on the catheter surface.

In addition to the fusion protein’s demonstrated antibacterial properties when bound to a surface, it also demonstrates anticoagulation properties. Heparin-binding peptides have proven efficacious in both soluble and immobilized forms being able to selectivity bind heparin from solutions [36]. For the fusion protein described here, the aPTT clotting assay confirmed the heparin capturing ability of the protein, since heparin from the sample is rinsed off before the assay. The uncoated samples were unable to capture and retain the heparin and hence showed the formation of clot.

However, these independent assays were not able to assess the ability of the coating to function as both and antimicrobial and anticoagulation. Thus, to evaluate simultaneous antibacterial and anticoagulation properties of the coating, an aPTT assay was conducted following incubation with bacteria, under the supposition that since the ATP assay previously established that the coating could prevent a biofilm, if the optical density of the clotting assay was lower it would indicate that the coating maintains its ability to function as an anticoagulation surface even after exposure to bacteria (Figure 9). As assumed, the coating retained it anticoagulant properties even after bacterial exposure confirming its simultaneous antimicrobial and anticoagulation properties. Three possible mechanisms for this simultaneous property are proposed; either the residues responsible for antimicrobial activity and anticoagulant properties were different or the amount of bacteria does not fully saturate the protein coating, or the bacteria disassociate from the coating, freeing up the necessary sites for heparin binding. In order to simulate in vivo blood flow conditions, we designed an in vitro flow model (Figure 2) that can provide shear stress seen in a catheter while it is inside the body. The flow parameters were calculated based on the dimensions of a typical hemodialysis catheter and the coated sheets were subjected to plasma flow under stress. These sheets were taken and assayed for antimicrobial and anticoagulant functions as described previously. The optical density of the coated sheets after exposure to plasma flow was less compared to the uncoated suggesting that the coating was able to sustain the shear stress of the flowing plasma and retained its properties.

Figure 2:

An in vitro flow system to simulate shear stress due to blood flow

Acronyms:

Silk₂: two units of silk gene sequence

S₄H₄: Four units of silk gene sequence and four units of heparin binding gene sequence

PBS: Phosphate buffer saline

CFU: Colony forming units

OD: Optical density

Conclusion

A genetically modified recombinant fusion protein consisting of a spider silk protein motif and a heparin-binding motif was cloned and expressed in bacteria. This fusion protein was successfully coated onto a silicone surface, which is a commonly used catheter material. Coating changed the hydrophobic silicone surface to a hydrophilic surface. The coating not only showed antimicrobial properties but also prevented the formation of biofilm. Finally, the coating proved to be both anti-coagulant and anti-infective simultaneously. This unique approach may prove effective to address and resolve many of the issues like infection, and thrombosis surrounding the use of blood-contacting medical devices.

Figure 1:

Graphical representation of Hypothesis. Spider silk is functionalized with heparin binding peptide (HBP) and coated on catheter tubing which can capture endogenous heparin to make it anti-infective and anti-thrombotic surface