J Pept Sci. 2017 Feb;23(2):162-171. doi: 10.1002/psc.2938.

Weighing biointeractions between fibrin(ogen) and clot-binding peptides using microcantilever sensors.

PubMed link

 

Supplement:

Background – Introduction

Cancer represents the leading cause of morbidity and mortality worldwide with 14 million new cases and 8.2 million cancer related deaths in 2012, the latter year in which information is available.1 Therefore, it requires detection at an early stage to increase the percentage of success of the oncological treatment.

Within the context of early cancer detection, tumor-homing peptides are promising agents to deliver imaging contrast and drugs to tumor sites due to their features2-3(i.e. improved tissue penetrating ability, low immunogenicity, high affinity to targets, stability, and easy of handling, among others), thus providing molecular and physiological information as well as enabling for individualized treatment.

Recently, we have satisfactorily reported the use of CREKA and CR(NMe)EKA (Scheme 1), which are linear pentapeptides recognizing clotted plasma proteins (fibrin-fibronectin complexes) in the blood vessels and stroma of tumors, in bioactive platforms.4-5 Specifically, these tumor-homing peptides were combined with poly(3,4-ethylenedioxythiophene) (PEDOT), a conducting polymer of excellent electrochemical properties, to render biocomposite films with enhanced electroactivity, electrostability, and electrical conductivity. However, not only did CR(NMe)EKA improved such features by acting as dopant agent and precluding the degradation of PEDOT, but also it was crucial to promote the adhesion of tumor cells onto fibrin-coated PEDOT-CR(NMe)EKA films. Indeed, the presence of the N-methyl-Glu residue has been shown to induce a better tumor-homing response in comparison to CREKA.

 

Scheme 1. Tumor-homing peptides used in this work: CR(NMe)EKA.

 

Nanomechanical sensing (Mecwins S.L.)

Therefore, taking into consideration the potential application of CREKA-based systems in the biotechnological field, the featured work was focused on understanding the biological interactions that CR(NMe)EKA establishes with clotted plasma proteins by means of nanomechanical sensing, a label-free sensing technology.6-7 Briefly, this technology is based on the microcantilever mechanical response after interacting with a biological analyte, either as a deformation-bending (static mode) or a resonance frequency shift (dynamic mode). Nanomechanical sensors display several advantages in comparison to established bioanalytical techniques (e.g. ELISA, microarrays and electrophoresis methods) such as manageability, easy of synthesis and functionalization, and high intrinsic sensitivity, that make them suitable for specific biomedical purposes: drug detection, quantification of biological agents, as well as understanding biological interactions, and detecting bacterial resistance to antibiotics. Accordingly, the fibrin(ogen) binding event was detected as the shift in the resonance frequency of microcantilevers in accordance to the adsorbed mass during dynamic mode testing.

 

Figure 1. Commercial Si chips used in this work containing arrays of 8 cantilevers (500 µm long, ~100 µm wide, and 1 µm thick).

 

Results

Specifically, CREKA and CR(NMe)EKA were covalently linked via an epoxysilane-based protocol to Si substrates that efficiently activated by UV/ozone treatment. Thus, to ensure an efficient surface immobilization, each step of the functionalization process was characterized by contact angle (CA), interferometry, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The results derived from the different characterization techniques performed at each one of the functionalization steps confirmed the suitability of the protocol to tether these linear, small pentapeptides onto Si surfaces.

Indeed, the wettability of the Si surface changed according to each step of the protocol. In the final step of the functionalization, the process induces a slight increase in the surface hydrophobicity regardless the pentapeptide used, CREKA (51.9° ± 3.9°) or CR(NMe)EKA (49.4° ± 4.5°). Although contact angle (CA) results are in good agreement with the expected chemical composition of each functionalization step, the functionalization protocol was corroborated by XPS. Furthermore, upon coating with the pentapeptide, the smooth and homogenous features of Si-OH (Rq = 0.4 nm) evolved towards a rougher surface, Rq was determined to be in the range between 0.4 nm and 1 nm. Moreover, although the presence of the NMe-Glu residue had no impact regarding the functionalization result, CR(NMe)EKA-functionalized silicon substrates yield the highest Fb adsorption in PBS in comparison to CREKA-functionalized surfaces. Hence, after the covalent binding of CR(NMe)EKA onto Si surfaces, NMe-Glu residue still promotes Fb-binding.

Finally, dynamic mode nanomechanical tests were carried out using CR(NMe)EKA-functionalized microcantilever sensors. This simple and manageable label-free detection technique provided information regarding the interaction between Fb/Fg and the clot-binding peptide, thus establishing a detection limit of 100 ng/mL. Specifically, CR(NMe)EKA-functionalized Si chips containing arrays of 8 cantilevers (Figure 1), thus using various sensors in parallel, were operated in the dynamic mode. After being cleaned, these commercial chips – 500 µm long, ~100 µm wide, and 1 µm thick – exhibited a fundamental resonance frequency of 5205.6 ± 161.7 kHz (n = 65) measured in a N2 atmosphere at 25 ºC. Once coated, CR(NMe)EKA biointeracts towards the two clotted plasma proteins, thus shifting the frequency as a consequence of Fb/Fg recognition (Scheme 2).

 

 

Scheme 2. Experimental set-up for the nanomechanical biosensing tests and schematics of the mechanical resonance frequency shift of a Si cantilever before and after the Fb recognition event (10 µg/mL in PBS).

 

Relevance – Impact

However, although further improvement is required to lower the detection limit and determine the specific binding affinity of CREKA and its analogues, the overall of the results obtained reflect the importance of developing emerging technologies suitable for specific biomedical purposes. In our opinion, the relevance of the work relies on the following aspects: our study combines peptide materials with an advanced nanotechnological tool for ultrasensitive detection of biological interactions. Thus, the application of this simple and manageable label-free detection technique provided information regarding the interaction between fibrin(ogen) and CR(NMe)EKA. Moreover, the functionalization protocol of the tumor-homing peptide onto silicon surfaces was conducted satisfactorily: the N-methyl-Glu residue, which is decisive to promote fibrin(ogen) binding, was not altered after the covalent linkage of the peptide to the surface.

Overall, even though the underlying binding mechanism of the tumor-homing peptide CR(NMe)EKA is complex and further work is required to determine its specific binding affinity, the featured study highlights the importance of developing emerging technologies suitable for specific biomedical purposes. We believe that this topic and our results, which contribute to the study of this tumor-homing pentapeptide as well as expanding the scope of nanomechanical biosensing, will be of high interest to the scientific community.

 

References

[1] Stewart B.W., Wild C. P. “World Cancer Report 2014,” Lyon, 2014.
[2] Li Z.J., Cho C.H. Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery. J. Transl. Med. 2012; 10(Suppl 1): S1.
[3] Hajitou A., Pasqualini R., Arap W. Vascular targeting: recent advances and therapeutic perspectives. Trends Cardiovasc. Med. 2006; 16: 80–88.
[4]  Fabregat G., Teixeira-Dias B., del Valle, L. J., Armelin E., Estrany F., Alemán C. ACS Appl. Mater Interfaces 2014; 6: 11940-11954.
[5] Puiggalí-Jou A., del Valle L. J., Armelin E., Alemán C. Macromol. Biosci. 2016; 16: 1461−1474.
[6] Shekhawat G.S., Dravid V.P. Biosensors: Microcantilevers to lift biomolecules. Nat. Nanotechnol. 2015; 10: 830–831.
[7] Calleja M., Kosaka P.M., San Paulo Á., Tamayo J. Challenges for nanomechanical sensors in biological detection. Nanoscale 2012; 4: 4925-4938.