Barbani, N.

Barcaro, G.

Bulgheresi, C.

Cristallini, C.

Giachino, Claudia

Monti, S.

Rizzo, A.

Rossin, Daniela

Rosso, Rachele

Sergi, F.

Villano, A.

English

Institutional Research Information System University of Turin

Abstract #962 ESB 2021Apr 12, 2021, 6:44:24 PM Page 1/4Abstract #962 | OralPredictive and experimental study ofintelligent nanoparticles for controllingcardiac ECM after myocardial infarctionG. Barcaro1, S. Monti2, A. Rizzo1, N. Barbani3, 1, A. Villano1, F. Sergi4, 3, C. Bulgheresi4, 3, R. Rosso4, D. Rossin4,C. Giachino4, C. Cristallini11 National Research Council (CNR), IPCF, Pisa, Italy2 National Research Council (CNR), ICCOM, Pisa, Italy3 University of Pisa, Department of Civil and Industrial Engineering, Pisa, Italy4 University of Turin, Department of Clinical and Biological Sciences, Turin, ItalyIntroductionRecent advancements in nanotechnology have the potential to revolutionize both preventive andtherapeutic approaches for treating cardiovascular disease [1]. Nanoengineering approaches could beexploited to develop scaffolds recapitulating the complex signals necessary for cardiac regeneration.Recently, cardiac scaffolds were modified with nanoparticles (NPs) able to recognize a specific ECMmetalloproteinase (MMP) with the aim to prevent left ventricular (LV) dysfunction after myocardialinfarction (MI) [2]. It is known that an increased LV myocardial MMP activity and selective upregulation ofMMPs in heart failure occur. The control of MMP activation and expression in the failing human LVmyocardium represents a significant therapeutic target for heart disease. Our idea was to exploit themolecular imprinting polymers (MIP) in order to restore the correct MMPs/TIMPs balance. In this work, wehave explored more in depth the potential of this technique through a wide scenario of evaluations fromthe computational study to the synthesis and physico-chemical, dimensional and biologicalcharacterization.Experimental MethodsMethacrylic acid (MAA), poly(ethylene glycol) ethyl ether methacrylate ((PEG)EEMA) as monomers andtrimethylolpropane methacrylate (TRIM) as cross-linker were used. Human MMP-9 was used as template.MD simulations were performed by using the AMBER16 software employing the General Amber Force Field(GAFF) for MAA and (PEG) EEMA species and the ff14SB Force Field for the MMP-9 protein. The structure ofisolated MMP-9 protein was extracted from PDB database. MIP NPs were synthesized using precipitationradical polymerisation of MAA and (PEG)EEMA with monomer ratio 75/25 (selected from the computationalanalysis) with a low coverage of the protein. FT-IR Chemical Imaging was used to evaluate the chemicalmap of pure MMP-9 and NP aggregates before and after template extraction. DLS analysis was carried outon different NP aqueous dispersions. SEM analysis was performed on both MIP NPs and control NPs. HPLCanalysis was performed to evaluate rebinding capacity and selectivity of MIP NPs towards MMP-9. Viabilityassay was carried out on H9c2 cardiomyoblasts incubated with increasing MIP NPs concentrations at 24, 48and 72 hrs through Propidium Iodide Flow Cytometry analysis.Results and DiscussionThe evolution of the interaction between MMP-9 and MIP NPs as a function of monomer concentration wasevaluated by a new and original computational protocol. A stronger water/protein interaction than themonomers/protein one was observed, although, in the case of low coverage, this effect was reducedespecially for (PEG)EEMA monomers that resulted more strongly anchored to the protein surface. FT-IRAbstract #962 ESB 2021Apr 12, 2021, 6:44:24 PM Page 2/4Chemical Imaging allowed to identify a diagnostic band for MMP-9 and detect this band both before andafter template extraction from MIP NPs. An interesting similarity between a partial coverage where theenzyme structure is exposed respect to monomer shell and chemical map showing a surface presence ofenzyme on NPs was observed. An high percentage of MMP-9 entrapped into NPs was evaluated by HPLCconfirming the strong interaction protein/NPs and an elevated enzyme extraction. SEM analysis showed anaverage value of sphere dimension only slightly higher for MIP NPs than control NPs. This result wasconfirmed by DLS analysis indicating that surface sites can take place allowing the removal of MMP-9 afterimprinting. HPLC analysis showed a rebinding capacity of MIP NPs towards the enzyme confirming thepresence of highly-specific recognition zones in agreement with the computational study.  MIP NPsexhibited also selectivity towards MMP-9 respect to an analogue enzyme (MMP-2). Finally, no cytotoxicactivity of MIP NPs was observed on H9c2 cells: the viability values were close to 100% independently ofdose and incubation time.ConclusionMolecular imprinting was proposed for the production of nanoparticles capable of modulating enzymes ofthe MMP family for restoring the correct MMPs regulation in the cardiac microenvironment. This study aimsto underline the importance of a comprehensive study, using a large and synergic set of analyses andmethodologies, to better explore and imitate the complex interactions between engineered nanosystemsand native tissue for progress in myocardial regeneration.AcknowledgementThis work was supported by transnational EU project INCIPIT M-ERA.NET 2 call 2016, MIUR FIRSTAffixReferences[1] Mahmoudi, M, Yu, M, Serpooshan, V, Wu, J.C., Langer, R, Lee , R.T., Karp , J.M., Farokhzad O.C.,2017, 'Multiscale technologies for treatment of ischemic cardiomyopathy', NatureNanotech 12, 845–855[2] Cristallini, C, Gagliardi, M, Barbani, N, Giannessi, D, Guerra, G.D., 2012, ‘Novel biodegradable,biomimetic and functionalised polymer scaffolds to prevent expansion of post-infarct left ventricularremodelling’, Journal of Materials Science: Materials in Medicine, 23:205-216Abstract #962 ESB 2021Apr 12, 2021, 6:44:24 PM Page 3/4SDF of monomers around the MMP-9Special distribution functions (SDF) of the MAA (yellow) and (PEG)EEMA (cyan) monomers around theMMP-9 protein in the presence of water.Abstract #962 ESB 2021Apr 12, 2021, 6:44:24 PM Page 4/4Rebinding MMP-9: MIP vs CPMMP-9 rebinding assay of MIP and control NPs (CP).

Predictive and experimental study of intelligent nanoparticles for controlling cardiac ECM after myocardial infarction

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Predictive and experimental study of intelligent nanoparticles for controlling cardiac ECM after myocardial infarction

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