SERS characterization of self-assembling oligopeptides for tissue engineering M. Di Foggia1, A. Torreggiani2, P. Taddei1, M. Dettin3, S. Sanchez-Cortes4, A. Tinti1 1 Dip. Scienze Biomediche e Neuromotorie, Università di Bologna, Italy. 2 ISOF-CNR, Bologna, Italy. 3 Dip. di Processi Chimici dell’Ingegneria, Università di Padova, Italy. 4 Dep. de Espectroscopia Vibracional y Procesos Multifotonicos, Madrid, Spain.

1st Faculty of Chemistry Bologna docet 1929 1st University 1st Faculty of Chemistry 1st Raman Spectrometer 1st SERS?

BIOMEDICAL MATERIALS SURFACE PROPERTIES Growth and support of damaged tissues Secret of success… SURFACE PROPERTIES improving the morphology of the material itself (porosity, synthesis, roughness…) inserting something that cells love!

SELF-ASSEMBLING PEPTIDES Biomimetic coatings for orthopaedics and dentistry Biocompatible and biodegradable Form membranes and stable fibrillar aggregates Many biomedical and pharmaceutical applications: drug-release systems matrices supporting cellular growth (neurite growth and synapsis formation) LEGO® peptides

leukocytes excrete high levels of ·OH: O2·- + H2O2 → ·OH + OH- + O2 The peptides “hide” the prosthetic device to the immunitary system, but the surgical procedure triggers the beginning of an inflammatory process: leukocytes excrete high levels of ·OH: O2·- + H2O2 → ·OH + OH- + O2 g-radiation of aqueous solutions of peptides in protected atmosphere (NO2), produces ·OH at physiological concentrations: H2O + hν → ·OH + H· Can radical attack alter peptide structure, thus hampering biomimetic coating functionality? 5

PEPTIDES EAK-161 + - b-sheet structure (best interaction surface-peptide-cells): electrostatic interactions between charged side chains (COO-/NH3+) hydrophobic interactions between aliphatic side chains p-p interaction between aromatic side chains 1 Zhang S et al, PNAS, 1993

PEPTIDES

STRUCTURE SENSITIVE BANDS Amides bands depends on H-bonds strenght: highly sensitive structural bands. Amide II Amide III Amide I Raman

METHODS Structural changes caused by radical attack were evaluated by FT-Raman spectroscopy and SERS technique on Ag colloids (≈ physiological conditions). SERS can be used to establish the relative importance of specific functional groups in controlling peptide adsorption (very debated problem!). Hydroxylamine hydrochloride Cl-

Radiation treatment: peptides 1, 3, 5, 7, 8 non-irradiated peptide difference spectrum

Peptides 2, 4: Asp  Glu non-irradiated peptide difference spectrum Structural variations on backbone chain Structural variations on aliphatic side-chains b-sheet decrease a-elix/random increase non-irradiated peptide difference spectrum

Structural variation in Tyr Peptide 6: Tyr Structural variation in Tyr side chains non-irradiated peptide difference spectrum

CONCLUSIONS (part 1) Peptides 2 and 4 showed severe structural variations: Asp decarboxylation Peptides 1, 3, 5, 7 and 8 were resistant to radical attack (1, 5 and 7 were found to stimulate osteoblasts proliferation in cell culture studies) 13

SERS spectra: peptides 1, 2, 7 Ag-COO- interaction ns COO- Am. III nas COO- b-sheet n C-COO- _ SERS _ FT-Raman Am. IV r CO

SERS spectra: peptides 3, 4 Mainly Orn (NH3+…Cl-) interaction r CH2 def as NH3+ t NH3+ Am. IV n COO- def NH3+ n C-COO- r NH3+ _ SERS _ FT-Raman Ag Cl- Cl-

Both COO- and NH3+ interaction SERS spectra: peptide 5 Both COO- and NH3+ interaction n COO- def as NH3+ n C-COO- _ SERS _ FT-Raman b-sheet Am. III

Both COO- and amide bond interaction SERS spectra: peptide 8 Both COO- and amide bond interaction Am. I n COO- n C-COO- Am. III _ SERS _ FT-Raman Am. IV

Both Tyr- and COO- interaction SERS spectra: peptide 6 Both Tyr- and COO- interaction 1605 Tyr- n COO- Tyr- Tyr- _ SERS _ FT-Raman Tyr- 1616 n C-COO-

Small changes in the SERS spectra SERS spectra: peptide 1, g-irradiation Small changes in the SERS spectra SERS Difference spectrum ns COO- Am. IV r CO n C-COO-

Major changes in the SERS spectra SERS spectra: peptide 4, g-irradiation Major changes in the SERS spectra ns COO- SERS Am. I Am. III n C-COO- def NH3+ Am. IV Difference spectrum def as NH3+ r CH2 t NH3+

CONCLUSIONS (part 2) SERS spectra evidence different peptide – colloid interactions as a function of a.a. substitution. SERS spectra highlights major changes on the peptide – colloid interaction for g-degraded peptides (i.e. pept 2, 4 and 6). Bibliography: [2] A. Tinti, M. Di Foggia, P. Taddei, A. Torreggiani, M. Dettin, C. Fagnano, J. Raman Spetrosc. 39 (2008) 250. [3] M. Di Foggia, P. Taddei, C. Fagnano, A. Torreggiani, M. Dettin, S. Sanchez-Cortes, A. Tinti, J. Mol. Struct. 924 (2009), 120. [4] M. Di Foggia, P. Taddei, A. Torreggiani, M. Dettin, A. Tinti, J. Raman Spetrosc. 42 (2011) 276. [5] M. Di Foggia, P. Taddei, A. Torreggiani, M. Dettin, A. Tinti, J. Raman Spetrosc. (2013) DOI 10.1002/jrs.4271., 21

Thank you for your kind attention! Ab-initio calculations: attributions interaction type Thank you for your kind attention! 22