Name: wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion
Uploaded: 2016-08-15T14:00:00.000+00:00
Duration: 6 min 15 s
Description: Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

1. Preparation of Sodium Naphthalenide Activating Solution and Surface Activation
Note: Carry out reactions in a well-ventilated fume hood. Follow general rules for handling highly flammable solvents and corrosive metals like metallic sodium. Naphthalene has a very unpleasant smell (mothball), even in very small amounts! If not indicated otherwise reactions are performed at room temperature. Sodium azide is highly toxic! THF (99.9%, see List of Materials) was stored over approximately 20% (by volume) molecular sieve. Distill THF with a noticeable water content over sodium. The formation of sodium naphthalenide does not occur if trace amounts of water are present.
<ol>
	<li>To a solution of 1.4 g (10.9 mmol) of naphthalene in 20 ml tetrahydrofuran (THF, dried over 3 &#197; molecular sieve), add 0.25 g (10.9 mmol) sodium metal in a screw-capped 100 ml glass bottle equipped with a PTFE-coated magnetic stirring bar. 
	Note: Dissolution can be greatly enhanced by cutting the sodium into small pieces and modest heating (35 - 40 &#176;C). The final solution has a dark, slightly greenish color and may be stored under strictly dry conditions.</li>
	<li>Punch out PTFE discs of 12 mm diameter from 0.5 mm thick foil material. Mark one side (e.g., using a letter stamp punch) and clean with iso-propanol.</li>
	<li>Incubate PTFE-samples individually in the activating solution (step 1.1) for 1 - 2 min using forceps. Note: The color change from white to dark brown indicates successful treatment.</li>
	<li>Subsequently rinse twice with THF and then with iso-propanol. 
	Note: The naphthalenide solution is exhausted when its color changes to a watery light brown. Unused naphthalenide solution (possibly containing metallic sodium) has to be decomposed by slowly adding iso-propanol prior to disposal.</li>
	<li>Oxidize treated samples in hydrogen peroxide (30%) containing 20% (w/v) trichloroacetic acid for 3 hr. Wash with water and dry. The surface now has a slight brownish appearance. Note: Activation and oxidation result in a drastically increased wettability of the surface. This finding was examined in detail previously 14.</li>
	<li>Treat oxidized discs with 50% (v/v) hexamethylene diisocyanate (HMDI) in dry THF for 2 hr, rinse with THF and leave to dry.</li>
	<li>Hydrolyze isocyanate bearing samples in water for 2 - 3 hr and dry. Note: The final amino-functionalized PTFE surface is far more stable than the isocyanate-bearing samples and is compatible with many standard crosslinkers for further coupling.</li>
</ol>
2. Peptide Immobilization
<ol>
	<li>Prepare a solution of 20% (v/v) diethyleneglycol diglycidyl ether (diepoxide) in 50 mM carbonate buffer, pH 9.</li>
	<li>Place the aminated discs with the marked side up individually into a 24 well plate and add 1.5 ml of the epoxide solution. Ensure full coverage of the samples. Incubate for 2 hr and wash two times with water and once with carbonate buffer.</li>
	<li>Add 50 &#181;l of a 0.5 mg/ml peptide (e.g., REDV) in 50 mM carbonate buffer, pH 9 containing 0.01% NaN3, to the bottom of individual wells of a fresh 24 well plate and carefully place the epoxy-functionalized discs upside-down (i.e., marked side down) onto the drop of the peptide solution. Make sure that the space between the bottom of the well and the PTFE disc is completely wetted due to capillary action.</li>
	<li>Incubate in a wet chamber (e.g., any sealable plastic box with a tight closing lid) with humidified atmosphere (by placing wet tissue paper on the bottom) for at least 3 hr or overnight. 
	Note: Albeit the REDV motif may be regarded as well-characterized, a scrambled or reverse amino acid sequence may be included as an additional negative control.</li>
	<li>Wash three times with water and sterilize in 50% iso-propanol/water for at least 30 min. Prior to cell seeding, rinse the samples in sterile phosphate buffered saline (PBS).</li>
</ol>
3. Cell Seeding
<ol>
	<li>Grow HUVECs using standard cell culture procedures 17,18.</li>
	<li>Place the peptide-conjugated samples with the modified side up in a 24 well plates. Use untreated PTFE discs as a control.</li>
	<li>Seed 5 x 104 HUVECs in 2 ml medium per well and incubate for 4 hr at 37 &#176;C and 5% CO2 in an incubator.</li>
	<li>Remove the discs, rinse carefully to remove unattached cells and transfer to a fresh 24 well plate. Add 2 ml of fresh medium and incubate for the desired period (e.g., 24 hr, 1 w, etc.). Change medium every two days.</li>
	<li>Prepare a stock solution of 1 mg/ml Calcein-AM in dimethyl sulfoxide (DMSO).</li>
	<li>After growing the cells remove the medium, wash with PBS and add PBS containing 1 &#181;l/ml Calcein-AM stain (e.g., 1 &#181;l of a stock solution per ml PBS). Gently agitate and incubate at 37 &#176;C for 45 min in the dark.</li>
	<li>Wash with PBS and immediately take micrographs on a fluorescence microscope equipped with standard FITC filters (Ex 488 nm, Em 515 nm). Use 100-fold magnification. Perform triplicates from unmodified as well as treated samples.</li>
	<li>Using ImageJ software determine the area colonized by the cells. Alternatively count the cells manually or use ImageJ. Both methods will give similar information about the attachment and growth of cells on the respective surfaces.</li>
</ol>

<ol>
	<li>Doctor, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+various+prosthetic+materials+and+newer+meshes+for+hernia+repairs.">Evaluation of various prosthetic materials and newer meshes for hernia repairs.</a> J. Minim. Access Surg. 2, 110-116 (2006).</li><li>Zaghal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+totally+implantable+venous+access+devices.">Update on totally implantable venous access devices.</a> Surg. Oncol. 21, 207-215 (2012).</li><li>Niu, G., Sapoznik, E., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+blood+vessels.">Bioengineered blood vessels.</a> Exp. Opin. Biol. Th. 14, 403-410 (2014).</li><li>Wang, M. -. J., Tsai, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomaterials in Blood-Contacting Devices: Complications and Solutions. , (2010).</li><li>de Mel, A., Jell, G., Stevens, M. M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalization+of+biomaterials+for+accelerated+in+situ+endothelialization:+a+review.">Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review.</a> Biomacromolecules. 9, 2969-2979 (2008).</li><li>Zdrahala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+caliber+vascular+grafts.+Part+I:+state+of+the+art.">Small caliber vascular grafts. Part I: state of the art.</a> J. Biomat. Appl. 10, 309-329 (1996).</li><li>Cleary, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+tissue+engineering:+the+next+generation.">Vascular tissue engineering: the next generation.</a> Trends Mol. Med. 18, 394-404 (2012).</li><li>Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+and+other+recognition+sequences+for+integrins.">RGD and other recognition sequences for integrins.</a> Annu. Rev. Dev. Bi. 12, 697-715 (1996).</li><li>Lei, Y., Remy, M., Labrugere, C., Durrieu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+immobilization+on+polyethylene+terephthalate+surfaces+to+study+specific+endothelial+cell+adhesion,+spreading+and+migration.">Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration.</a> J. Mat. Sci. Mater. M. 23, 2761-2772 (2012).</li><li>Gabriel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+RGD+Modification+of+the+Inner+Pore+Surface+of+Polycaprolactone+Scaffolds.">Covalent RGD Modification of the Inner Pore Surface of Polycaprolactone Scaffolds.</a> J. Biomat. Sci.. Polym. E. 23, 941-953 (2012).</li><li>Ceylan, H., Tekinay, A. B., Guler, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adhesion+and+growth+of+vascular+endothelial+cells+on+bioactive+peptide+nanofiber+functionalized+stainless+steel+surface.">Selective adhesion and growth of vascular endothelial cells on bioactive peptide nanofiber functionalized stainless steel surface.</a> Biomaterials. 32, 8797-8805 (2011).</li><li>Chlupac, J., Filova, E., Bacakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+replacement:+50+years+of+development+and+tissue+engineering+paradigms+in+vascular+surgery.">Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery.</a> Physiol. Res. / Academia Scientiarum Bohemoslovaca. 58, 119-139 (2009).</li><li>Wise, S. G., Waterhouse, A., Kondyurin, A., Bilek, M. M., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-based+biofunctionalization+of+vascular+implants.">Plasma-based biofunctionalization of vascular implants.</a> Nanomedicine UK. 7, 1907-1916 (2012).</li><li>Mikulikova, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+microarrays+on+photochemically+modified+polytetrafluoroethylene.">Cell microarrays on photochemically modified polytetrafluoroethylene.</a> Biomaterials. 26, 5572-5580 (2005).</li><li>Gabriel, M., Dahm, M., Vahl, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wet-chemical+approach+for+the+cell-adhesive+modification+of+polytetrafluoroethylene.">Wet-chemical approach for the cell-adhesive modification of polytetrafluoroethylene.</a> Biomed. Mater. 6, 035007 (2011).</li><li>Gabriel, M., van Nieuw Amerongen, G. P., Van Hinsbergh, V. W., Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+grafting+of+RGD-motif-containing+peptide+on+the+surface+of+polycaprolactone+films.">Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films.</a> J. Biomat. Sci.. Polym. E. 17, 567-577 (2006).</li><li>Larsen, C. C., Kligman, F., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+RGD+fluorosurfactant+polymer+modification+of+ePTFE+on+endothelial+cell+adhesion,+growth,+and+function.">The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.</a> Biomaterials. 27, 4846-4855 (2006).</li><li>Gabriel, M., Nazmi, K., Veerman, E. C., Nieuw Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+LL-37-grafted+titanium+surfaces+with+bactericidal+activity.">Preparation of LL-37-grafted titanium surfaces with bactericidal activity.</a> Bioconjugate Chem. 17, 548-550 (2006).</li><li>Lotz, A., Heller, M., Brieger, J., Gabriel, M., F&#246;rch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Plasma+Polymerized+Thin+Films+and+Attachment+of+Biomolecules+to+Influence+HUVEC-Cell+Adhesion.">Derivatization of Plasma Polymerized Thin Films and Attachment of Biomolecules to Influence HUVEC-Cell Adhesion.</a> Plasma Process Polym. 9, 10-16 (2012).</li></ol>

The authors have nothing to disclose.

The detailed description of surface modification protocol of PTFE consists of successive steps starting with the elimination of fluorine from the polymer backbone as depicted in Figure 6. As a result, a layer is formed that contains an abundant amount of conjugated carbon-carbon double bonds in accordance with the dark brownish color that developed upon naphthalenide treatment. Standard oxidation with acidic hydrogen peroxide yields a hydroxylated surface accompanied by brightening to a pale brown, this reflects the loss of double bonds. This process is clearly evidenced by IR spectroscopy.
In order to achieve a more generally applicable functionality, the pendant OH-groups were converted into amino-functionalities by simple treatment with a diisocyanate and subsequent hydrolysis of the NCO-moieties to primary amines. This is favorable, because amine-bearing material can be stored for prolonged periods whereas the highly reactive isocyanate is susceptible to the very presence of air humidity.
Care must be taken in the avoidance of even trace amounts of water in the initial activation step and the HMDI treatment because both sodium naphthalenide and the isocyanate are highly susceptible to hydrolysis. In contrast to standard procedures in which the sample is completely immersed in the solution, the present protocol necessitates only small amounts of the reagent. Therefore this method saves a lot of the costly peptide.
Amine-bearing surfaces are ideal starting points for the coupling of many biomolecules. The diepoxide employed in this work is interesting in several regards: it is comparatively cheap, hydrophilic, highly reactive towards amines and non-reacted epoxides are hydrolyzed in an aqueous environment into &#34;physiologically inert&#34; diols. Homobifunctional crosslinkers are suitable reagents where only one reactive group is present on the peptide to be immobilized. This applies for REDV containing only one single primary amino-group on the N-terminal (this is the same for the frequently used sequence RGDS). For peptides containing lysine residues (with side chain amines) the outcome of the conjugation is ambiguous, meaning that proper coupling does not occur in a defined way. In this case, a strategy using heterobifunctional crosslinkers (e.g., amine- and thiol-reactive) and cysteine (N- or C-terminal) containing peptides is helpful 18.
The results of the peptide immobilization in terms of cell adhesion is unambiguous (Figures 3 and 4). Whereas virtually no cells adhere on bare PTFE, REDV-modification renders the material an excellent cell-adhesive substrate. Complementary to the above mentioned protocol preliminary work show that this technique is also applicable on porous (expanded) PTFE with similar results (Figure 5). This is of special interest because this is the material vascular grafts are made of. In contrast to surface techniques that mostly make use of plasma treatment (plasma activation 13, plasma polymerization 19), this protocol provides a guideline for the covalent modification of PTFE by employing readily available chemicals and lab procedures and by avoiding highly sophisticated equipment. Plasma methodologies such as reactive plasma (O2, NH3, etc.) and plasma polymerization are not applicable within porous constructs or tubing because reactive species (e.g., radicals) are not able to penetrate these structures due to inactivation upon contact with the materials surface. For example small caliber vascular grafts (diameter &#60; 5 mm) cannot be treated in this way. In contrast to this, by means of wet chemistry, there is no restriction regarding the shape of the device. In this way, by enabling the formation of an endothelial layer on the luminal side of an ePTFE vascular graft, thrombogenicity and long term patency may be improved. In addition, a cell adhesive surface for surgical hernia meshes is supposed to result in better tissue integration.

Various polymers used in medicine that have been approved for some time do not exhibit enhanced biocompatibility, i.e., lack of cell-adhesiveness, induction of fibrotic encapsulation and thrombogenicity, to mention a few. Interactions between the biomaterial and the biological system takes place mainly at the surface of the implant. As a consequence, research has focused on surface modification in order to create appropriate properties for a desired application while leaving the bulk properties of the material unaffected. Polytetrafluoroethylene (PTFE) as a physiologically inert polymer is used in many medical fields such as hernia surgical mesh 1, medical ports 2 and, most importantly, vascular grafts 3.
Especially in blood contacting situations the hydrophobic nature of PTFE causes unspecific adsorption of plasma components and as a consequence platelet adhesion, often resulting in thrombotic events and occlusion of the graft 4. Furthermore, PTFE, like most polymers, does not support cellular adhesion and coverage which would be a desirable feature to induce the formation of a beneficial layer of endothelial cells (ECs) on the inner (luminal) surface of the vascular graft 5. A biomimetic endothelium is expected to fulfil many of the functions of its natural equivalent, notably its antithrombogenic properties 6. A general biomimetic modification strategy is based upon the concept of exclusively endowing the material with cell-adhesiveness while leaving the materials bulk properties unaffected. In addition, platelet adhesion may be reduced by incorporating anti-adhesive (anti-fouling) attributes 7. Various peptides &#8212; mostly derived from proteins of the extracellular matrix &#8212; have been described that strongly enhance cell-adhesion by binding to cellular receptors, belonging to the class of integrins 8. The best-known example in this regard is the peptide Arg-Gly-Asp (RGD) that interacts with most cell types. Other amino acid sequences are recognized by integrins exclusively expressed on specific cells. For example, Arg-Glu-Asp-Val (REDV) and Tyr-Ile-Gly-Ser-Arg (YIGSR) have been found to bind to ECs in a specific manner 9. Covalent immobilization of such peptides has been carried out on a plethora of inherently non-adhesive materials including metals and polymers 10,11.
Porous PTFE, more precisely expanded PTFE (ePTFE) &#8212; along with polyethylene terephthalate (PET) - is the most important material for the production of vascular grafts 12. Established physical techniques for appropriate treatments, such as plasma modification 13 or by photochemical methods 14, are hampered by the fact that porous and/or tubular structures are not readily treatable inside the pores or the lumen respectively. Wet chemistry on PTFE is a difficult task because of the highly inert nature of the fluorin-containing polymer that resists most chemical attacks 15.
In this paper we describe a comparatively facile method for a covalent modification strategy. Adapted from a procedure to render PTFE bondable, functional groups were created on the materials surface that serve as anchor points for further conjugation of biologically active molecules.

<table><tbody><tr><td>PTFE foil 0.5 mm</td><td>Cadillac Plastic&amp;nbsp;</td><td>n/a</td><td></td></tr><tr><td>REDV peptide</td><td>Genecust</td><td>n/a</td><td>custom synthesis &gt;95% purity</td></tr><tr><td>iso-propanol</td><td>Sigma Aldrich</td><td>34965</td><td></td></tr><tr><td>tetrahydrofurane (THF)</td><td>Sigma Aldrich</td><td>401757</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Sigma Aldrich</td><td>D8418</td><td></td></tr><tr><td>molecular sieve 3 &amp;Aring;</td><td>Sigma Aldrich</td><td>208574</td><td></td></tr><tr><td>sodium metal</td><td>Sigma Aldrich</td><td>483745</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>naphthalene</td><td>Sigma Aldrich</td><td>147141</td><td></td></tr><tr><td>hydrogen peroxide 30%</td><td>Sigma Aldrich</td><td>95321</td><td></td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399&amp;nbsp;</td><td></td></tr><tr><td>diethylene glycol diglycidyl ether</td><td>Sigma Aldrich</td><td>17741</td><td></td></tr><tr><td>hexamethylene diisocyanate (HMDI)</td><td>Sigma Aldrich</td><td>52650</td><td></td></tr><tr><td>Calcein-AM</td><td>Sigma Aldrich</td><td>56496</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014&amp;nbsp;</td><td></td></tr><tr><td>sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td></td></tr><tr><td>24 well plates</td><td>Greiner-Bio-One</td><td>662 160</td><td></td></tr><tr><td>ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td><td>&amp;nbsp;Nicolet</td><td>n/a</td><td></td></tr><tr><td>fluorescence microscope Olympus X-70</td><td>Olympus</td><td>n/a</td><td></td></tr><tr><td>humbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td>n/a</td><td></td></tr><tr><td>ePTFE vascular graft</td><td>Gore</td><td>n/a</td><td></td></tr></tbody></table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

The results of the crucial chemical reaction steps were monitored by IR spectroscopy (Figure 1). The initial activation with sodium naphthalenide generates double bonds &#8212; and to a minor extent &#8212; OH-functionalities. The signal indicating C=C bonds disappear upon oxidation, yielding a surface bearing almost exclusively hydroxyl-groups. Analysis of further standard conjugation steps are not shown here. The color changes due to activation and oxidation are in agreement with the expected chemistry that is used: conjugated double bonding systems are expected to be brownish and its loss results in brightening (Figure 2). In addition, the possible outcome of activation and oxidation on the surface morphology was investigated by means of scanning electron microscopy. Virtually no detrimental effect of the treatment was observed (Figure 2).
Figures 3 and 4 show the outcome of REDV-immobilization on endothelial cell growth. Whereas virtually no cell adhesion and proliferation occurs on untreated material the modification strongly supports colonization over a two week period. Exemplified for a clinical application (i.e., vascular grafts), the modification was identically performed on original material from a commercially available graft made of expanded PTFE with a similar results over a period of one week (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/54272/54272fig1.jpg" /> 
Figure 1. IR spectroscopy of PTFE. Treatment of pristine PTFE (A) results in the formation of double bonds and to a certain extent of hydroxy functions (B). Subsequently C=C bonds are reduced due to the oxidation (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54272/54272fig2.jpg" /> 
Figure 2. Optical appearance of bare and surface activated PTFE. (A) Whereas untreated PTFE (left) appears white, the activation using sodium naphthalenide yields a dark brownish color (middle) which is slightly brightened upon oxidation (right). Untreated (B) and oxidized PTFE (C) samples were additionally investigated using scanning electron microscopy (magnification: 2,000X). Discs are 12 mm in diameter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54272/54272fig3.jpg" /> 
Figure 3. Endothelial cell culture on pristine and peptide-modified PTFE. Untreated samples are not colonized by ECs (A, C, E after 24 hr, 1 w and 2 w respectively) whereas adhesion and growth on peptide-modified material is greatly enhanced (B, D and F after 24 hr, 1 w and 2 w respectively). Scale bar: 100 &#181;m, magnification 100X. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54272/54272fig4.jpg" /> 
Figure 4. Quantification of cell coverage by ImageJ analysis. Cellular growth on bare PTFE (A, C, E) and on REDV-conjugated polymer surfaces (B, D, F) expressed as percentage coverage of total area. Immobilized peptide clearly enables initial adhesion (B) and supports colonization over a 2-week period (D, F). Triplicate determinations, mean &#177; standard deviation). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54272/54272fig5.jpg" /> 
Figure 5. Endothelial cells grown for 1 week on expanded PTFE. The structure of ePTFE is shown using Scanning Electron Microscopy (A). The results obtained on porous material are in accordance with those obtained for flat PTFE specimen. In contrast to the few cells found on bare material (B) the modified surface (C) provides an excellent substrate for cell growth. Scale bars are 100 &#181;m for A, B and C respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54272/54272fig6.jpg" /> 
Figure 6. Schematic representation of the chemical modification of PTFE using wet-chemistry. Double bonds generated by Na-naphthalenide treatment are oxidized resulting in OH-functionalities. A hydroxyl-reactive diisocyanate is then immobilized and hydrolyzed to an amine. Finally the bifunctional diepoxide is applied to conjugate the REDV peptide using the N-terminal amino group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

wet-chemistry-peptide-immobilization-on-polytetrafluoroethylene-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.6K Views.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Video: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...