Albumin/Mitotane Interaction Affects Drug Activity in Adrenocortical Carcinoma Cells: Smoke and Mirrors on Mitotane Effect with Possible Implications for Patients' Management.

BACKGROUND: Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). Although it has been used for many years, its mechanism of action remains elusive. H295R cells are, in ACC, an essential tool to evaluate drug mechanisms, although they often lead to conflicting results. METHODS: Using different in vitro biomolecular technologies and biochemical/biophysical experiments, we evaluated how the presence of "confounding factors" in culture media and patient sera could reduce the pharmacological effect of mitotane and its metabolites. RESULTS: We discovered that albumin, the most abundant protein in the blood, was able to bind mitotane. This interaction altered the effect of the drug by blocking its biological activity. This blocking effect was independent of the albumin source or methodology used and altered the assessment of drug sensitivity of the cell lines. CONCLUSIONS: In conclusion, we have for the first time demonstrated that albumin does not only act as an inert drug carrier when mitotane or its metabolites are present. Indeed, our experiments clearly indicated that both albumin and human serum were able to suppress the pharmacological effect of mitotane in vitro. These experiments could represent a first step towards the individualization of mitotane treatment in this rare tumor.

Apelin-13 Increases Functional Connexin-43 through Autophagy Inhibition via AKT/mTOR Pathway in the Non-Myocytic Cell Population of the Heart.

Studies have shown a link between the downregulation of connexin 43 (Cx43), the predominant isoform in cardiac gap junctions, and high susceptibility to cardiac arrhythmias and cardiomyocyte death. Non-myocytic cells (NMCs), the most abundant component of the heart, exert multiple cardiac functions and represent an important therapeutic target for diseased cardiac tissue. A few studies have investigated the effect of Apelin-13, an endogenous peptide with a key role in various cardiovascular functions, on Cx43 expression in cardiomyocytes. However, it remained unknown whether Apelin-13 influences Cx43 expression in NMCs. Here, we found that in NMCs, Cx43 protein expression increased after Apelin-13 treatment (100 nM for 48 h). Furthermore, dye transfer assays proved that Apelin-13-treated NMCs had a greater ability to communicate with surrounding cardiomyocytes, and this effect was abrogated by carbenoxolone, a gap junction inhibitor. Interestingly, we showed that Apelin-13 increased Cx43 through autophagy inhibition, as proved by the upregulation of p62 and LC3I, acting as 3-MA, a well-known autophagy inhibitor. In addition, Apelin-13-induced AKT and mTOR phosphorylation was abolished by LY294002 and rapamycin inhibitors resulting in Cx43 increased suppression. These results open the possibility of targeting gap junctions in NMCs with Apelin-13 as an exciting therapeutic approach with great potential.

Therapeutic Acellular Scaffolds for Limiting Left Ventricular Remodelling-Current Status and Future Directions.

Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Following MI, the hypoxic microenvironment triggers apoptosis, disrupts the extracellular matrix and forms a non-functional scar that leads towards adverse left ventricular (LV) remodelling. If left untreated this eventually leads to heart failure. Besides extensive advancement in medical therapy, complete functional recovery is never accomplished, as the heart possesses limited regenerative ability. In recent decades, the focus has shifted towards tissue engineering and regenerative strategies that provide an attractive option to improve cardiac regeneration, limit adverse LV remodelling and restore function in an infarcted heart. Acellular scaffolds possess attractive features that have made them a promising therapeutic candidate. Their application in infarcted areas has been shown to improve LV remodelling and enhance functional recovery in post-MI hearts. This review will summarise the updates on acellular scaffolds developed and tested in pre-clinical and clinical scenarios in the past five years with a focus on their ability to overcome damage caused by MI. It will also describe how acellular scaffolds alone or in combination with biomolecules have been employed for MI treatment. A better understanding of acellular scaffolds potentialities may guide the development of customised and optimised therapeutic strategies for MI treatment.

Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions.

In the last decade, many papers highlighted that the histone variant H2AX and its phosphorylation on Ser 139 (γH2AX) cannot be simply considered a specific DNA double-strand-break (DSB) marker with a role restricted to the DNA damage response, but rather as a 'protagonist' in different scenarios. This review will present and discuss an up-to-date view regarding the 'non-canonical' H2AX roles, focusing in particular on possible functional and structural parts in contexts different from the canonical DNA DSB response. We will present aspects concerning sex chromosome inactivation in male germ cells, X inactivation in female somatic cells and mitosis, but will also focus on the more recent studies regarding embryonic and neural stem cell development, asymmetric sister chromosome segregation in stem cells and cellular senescence maintenance. We will discuss whether in these new contexts there might be a relation with the canonical DNA DSB signalling function that could justify γH2AX formation. The authors will emphasize that, just as H2AX phosphorylation signals chromatin alteration and serves the canonical function of recruiting DSB repair factors, so the modification of H2AX in contexts other than the DNA damage response may contribute towards creating a specific chromatin structure frame allowing 'non-canonical' functions to be carried out in different cell types.

Induced Pluripotent Stem Cells: Advances in the Quest for Genetic Stability during Reprogramming Process.

Evaluation of the extent and nature of induced pluripotent stem cell (iPSC) genetic instability is important for both basic research and future clinical use. As previously demonstrated regarding embryonic stem cells, such DNA aberrations might affect the differentiation capacity of the cells and increase their tumorigenicity. Here, we first focus on the contribution of multiple DNA damage response pathways during cellular reprogramming. We then discuss the origin and mechanisms responsible for the modification of genetic material in iPSCs (pre-existing variations in somatic cells, mutations induced by reprogramming factors, and mutations induced by culture expansion) and deepen the possible functional consequences of genetic variations in these cells. Lastly, we present some recent improvements of iPSC generation methods aimed at obtaining cells with fewer genetic variations.

H2AX phosphorylation level in peripheral blood mononuclear cells as an event-free survival predictor for bladder cancer.

Bladder cancer (BC) has a typical aetiology characterized by a multistep carcinogenesis due to environmental exposures, genetic susceptibility, and their interaction. Several lines of evidence suggest that DNA repair plays a role in the development and progression of BC. In particular, the study of individual susceptibility to DNA double strand breaks (DSBs) may provide valuable information on BC risk, and help to identify those patients at high-risk of either recurrence or progression of the disease, possibly personalizing both surveillance and treatment. Among the different DSB markers, the most well characterized is phosphorylation of the histone H2AX (γ-H2AX). We assessed any potential role of γ-H2AX as a molecular biomarker in a case-control study (146 cases and 146 controls) to identify individuals with increased BC risk and at high-risk of disease recurrence or progression. We investigated γ-H2AX levels in peripheral blood mononuclear cells before and after their exposure to ionizing radiation (IR). We did not find any significant difference among cases and controls. However, we observed a significant association between γ-H2AX basal levels and risk of disease recurrence or progression. In particular, both BC patients as a whole and the subgroup of non-muscle invasive BC (NMIBC) with high basal H2AX phosphorylation levels had a decreased risk of recurrence or progression (for all BC HR 0.70, 95%CI 0.52-0.94, P = 0.02; for NMIBC HR 0.68, 95%CI 0.50-0.92, P = 0.01), suggesting a protective effect of basal DSB signaling. Our data suggest that γ-H2AX can be considered as a potential molecular biomarker to identify patients with a higher risk of BC recurrence. © 2015 Wiley Periodicals, Inc.

Senescence in Human Mesenchymal Stem Cells: Functional Changes and Implications in Stem Cell-Based Therapy.

Regenerative medicine is extensively interested in developing cell therapies using mesenchymal stem cells (MSCs), with applications to several aging-associated diseases. For successful therapies, a substantial number of cells are needed, requiring extensive ex vivo cell expansion. However, MSC proliferation is limited and it is quite likely that long-term culture evokes continuous changes in MSCs. Therefore, a substantial proportion of cells may undergo senescence. In the present review, we will first present the phenotypic characterization of senescent human MSCs (hMSCs) and their possible consequent functional alterations. The accumulation of oxidative stress and dysregulation of key differentiation regulatory factors determine decreased differentiation potential of senescent hMSCs. Senescent hMSCs also show a marked impairment in their migratory and homing ability. Finally, many factors present in the secretome of senescent hMSCs are able to exacerbate the inflammatory response at a systemic level, decreasing the immune modulation activity of hMSCs and promoting either proliferation or migration of cancer cells. Considering the deleterious effects that these changes could evoke, it would appear of primary importance to monitor the occurrence of senescent phenotype in clinically expanded hMSCs and to evaluate possible ways to prevent in vitro MSC senescence. An updated critical presentation of the possible strategies for in vitro senescence monitoring and prevention constitutes the second part of this review. Understanding the mechanisms that drive toward hMSC growth arrest and evaluating how to counteract these for preserving a functional stem cell pool is of fundamental importance for the development of efficient cell-based therapeutic approaches.

Persistent DNA damage-induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated ß-galactosidase activity and enlarged γH2AX foci co-localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence-associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour-promoting behaviour.

Factors Ruling the Uptake of Silica Nanoparticles by Mesenchymal Stem Cells: Agglomeration Versus Dispersions, Absence Versus Presence of Serum Proteins.

The results of a systematic investigation of the role of serum proteins on the interaction of silica nanoparticles (NP) doped in their bulk with fluorescent molecules (IRIS Dots, 50 nm in size), with human mesenchymal stem cells (hMSCs) are reported. The suspension of IRIS Dots in bare Dulbecco-modified Eagle's medium results in the formation of large agglomerates (≈1.5 µm, by dynamic light scattering), which become progressively smaller, down to ≈300 nm in size, by progressively increasing the fetal bovine serum (FBS) content of the solutions along the series 1.0%, 2.5%, 6.0%, and 10.0% v/v. Such difference in NP dispersion is maintained in the external cellular microenvironment, as observed by confocal microscopy and transmission electron microscopy. As a consequence of the limited diffusion of proteins in the inter-NP spaces, the surface of NP agglomerates is coated by a protein corona independently of the agglomerate size/FBS concentration conditions (ζ-potential and UV circular dichroism measurements). The protein corona appears not to be particularly relevant for the uptake of IRIS Dots by hMSCs, whereas the main role in determining the internalization rate is played by the absence/presence of serum proteins in the extracellular media.

Human mesenchymal stem cells labelled with dye-loaded amorphous silica nanoparticles: long-term biosafety, stemness preservation and traceability in the beating heart.

BACKGROUND: Treatment of myocardial infarction with mesenchymal stem cells (MSCs) has proven beneficial effects in both animal and clinical studies. Engineered silica nanoparticles (SiO2-NPs) have been extensively used as contrast agents in regenerative medicine, due to their resistance to degradation and ease of functionalization. However, there are still controversies on their effective biosafety on cellular systems. In this perspective, the aims of the present study are: 1) to deeply investigate the impact of amorphous 50 nm SiO2-NPs on viability and function of human bone marrow-derived MSCs (hMSCs); 2) to optimize a protocol of harmless hMSCs labelling and test its feasibility in a beating heart model. RESULTS: Optimal cell labelling is obtained after 16 h exposure of hMSCs to fluorescent 50 nm SiO2-NPs (50 µg mL(-1)); interestingly, lysosomal activation consequent to NPs storage is not associated to oxidative stress. During prolonged culture hMSCs do not undergo cyto- or genotoxicity, preserve their proliferative potential and their stemness/differentiation properties. Finally, the bright fluorescence emitted by internalized SiO2-NPs allows both clear visualization of hMSCs in normal and infarcted rat hearts and ultrastructural analysis of cell engraftment inside myocardial tissue. CONCLUSIONS: Overall, 50 nm SiO2-NPs display elevated compatibility with hMSCs in terms of lack of cyto- and genotoxicity and maintenance of important features of these cells. The demonstrated biosafety, combined with proper cell labelling and visualization in histological sections, make these SiO2-NPs optimal candidates for the purpose of stem cell tracking inside heart tissue.

High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells.

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

Effect of blood storage conditions on DNA repair capacity measurements in peripheral blood mononuclear cells.

Due to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported. These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.

Maintenance of genomic stability in mouse embryonic stem cells: relevance in aging and disease.

Recent studies have shown that mouse embryonic stem cells (mESCs) rely on a distinctive genome caretaking network. In this review, we will discuss how mESCs functionally respond to DNA damage and describe several modifications in mESC DNA damage response, which accommodate dynamic cycling and preservation of genetic information. Subsequently, we will discuss how the transition from mESCs to adult stem/progenitor cells can be involved in the decline of tissue integrity and function in the elderly.

Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration.

Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.

APJ as Promising Therapeutic Target of Peptide Analogues in Myocardial Infarction- and Hypertension-Induced Heart Failure.

The widely expressed G protein-coupled apelin receptor (APJ) is activated by two bioactive endogenous peptides, apelin and ELABELA (ELA). The apelin/ELA-APJ-related pathway has been found involved in the regulation of many physiological and pathological cardiovascular processes. Increasing studies are deepening the role of the APJ pathway in limiting hypertension and myocardial ischaemia, thus reducing cardiac fibrosis and adverse tissue remodelling, outlining APJ regulation as a potential therapeutic target for heart failure prevention. However, the low plasma half-life of native apelin and ELABELA isoforms lowered their potential for pharmacological applications. In recent years, many research groups focused their attention on studying how APJ ligand modifications could affect receptor structure and dynamics as well as its downstream signalling. This review summarises the novel insights regarding the role of APJ-related pathways in myocardial infarction and hypertension. Furthermore, recent progress in designing synthetic compounds or analogues of APJ ligands able to fully activate the apelinergic pathway is reported. Determining how to exogenously regulate the APJ activation could help to outline a promising therapy for cardiac diseases.

Mediating Role of Lifestyle Behaviors in the Association between Education and Cancer: Results from the European Prospective Investigation into Cancer and Nutrition.

BACKGROUND: Many studies have shown that socioeconomic position (SEP) is associated with the incidence of malignant tumors at different sites. This study aims to estimate the association between educational level (as proxy for SEP) and cancer incidence and to understand whether the observed associations might be partially explained by lifestyle behaviors. METHODS: The analyses were performed on data from the European Prospective Investigation into Cancer and Nutrition (EPIC) study, globally and by sex. We used Cox proportional hazards models together with mediation analysis to disentangle the total effect (TE) of educational level [measured through the Relative Index of Inequality (RII)] on cancer incidence into pure direct (PDE) and total indirect (TIE) effect, unexplained and explained by mediators, respectively. PDE and TIE were then combined to compute the proportions mediated (PM). RESULTS: After an average of 14 years of follow-up, 52,422 malignant tumors were ascertained. Low educated participants showed higher risk of developing stomach, lung, kidney (in women), and bladder (in men) cancers, and, conversely, lower risk of melanoma and breast cancer (in post-menopausal women), when compared with more educated participants. Mediation analyses showed that portions of the TE of RII on cancer could be explained by site-specific related lifestyle behaviors for stomach, lung, and breast (in women). CONCLUSIONS: Cancer incidence in Europe is determined at least in part by a socioeconomically stratified distribution of risk factors. IMPACT: These observational findings support policies to reduce cancer occurrence by altering mediators, such as lifestyle behaviors, particularly focusing on underprivileged strata of the population.

Fluorescent silica nanoparticles improve optical imaging of stem cells allowing direct discrimination between live and early-stage apoptotic cells.

Highly bright and photostable cyanine dye-doped silica nanoparticles, IRIS Dots, are developed, which can efficiently label human mesenchymal stem cells (hMSCs). The application procedure used to label hMSCs is fast (2 h), the concentration of IRIS Dots for efficient labeling is low (20 µg mL(-1) ), and the labeled cells can be visualized by flow cytometry, confocal microscopy, and transmission electron microscopy. Labeled hMSCs are unaffected in their viability and proliferation, as well as stemness surface marker expression and differentiation capability into osteocytes. Moreover, this is the first report that shows nonfunctionalized IRIS Dots can discriminate between live and early-stage apoptotic stem cells (both mesenchymal and embryonic) through a distinct external cell surface distribution. On the basis of biocompatibility, efficient labeling, and apoptotic discrimination potential, it is suggested that IRIS Dots can serve as a promising stem cell tracking agent.

Role of Chaperone-Mediated Autophagy in Ageing Biology and Rejuvenation of Stem Cells.

What lies at the basis of the mechanisms that regulate the maintenance and self-renewal of pluripotent stem cells is still an open question. The control of stemness derives from a fine regulation between transcriptional and metabolic factors. In the last years, an emerging topic has concerned the involvement of Chaperone-Mediated Autophagy (CMA) as a key mechanism in stem cell pluripotency control acting as a bridge between epigenetic, transcriptional and differentiation regulation. This review aims to clarify this new and not yet well-explored horizon discussing the recent studies regarding the CMA impact on embryonic, mesenchymal, and haematopoietic stem cells. The review will discuss how CMA influences embryonic stem cell activity promoting self-renewal or differentiation, its involvement in maintaining haematopoietic stem cell function by increasing their functionality during the normal ageing process and its effects on mesenchymal stem cells, in which modulation of CMA regulates immunosuppressive and differentiation properties. Finally, the importance of these new discoveries and their relevance for regenerative medicine applications, from transplantation to cell rejuvenation, will be addressed.

Silica Nanoparticle Internalization Improves Chemotactic Behaviour of Human Mesenchymal Stem Cells Acting on the SDF1α/CXCR4 Axis.

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo.

Molecularly Imprinted Nanoparticles towards MMP9 for Controlling Cardiac ECM after Myocardial Infarction: A Predictive Experimental-Computational Chemistry Investigation.

The recent advances in nanotechnology are revolutionizing preventive and therapeutic approaches to treating cardiovascular diseases. Controlling the extracellular matrix metalloproteinase (MMP) activation and expression in the failing human left ventricular myocardium represents a significant therapeutic target for heart disease. In this study, we used molecularly imprinting polymers (MIPs) to restore the correct balance between MMPs and their tissue inhibitors (TIMPs), and explored the potential of this technique exhaustively through chemical synthesis, physicochemical and biological characterizations, and computational chemistry methods. By molecular dynamics simulations based on classical force fields, we simulated the early stages of the imprinting process in solution disclosing the pivotal interaction established between the monomers and the MMP9 protein template. The average interaction energies of methacrylic acid (MAA) and poly (ethylene glycol) ethyl ether methacrylate (PEG) units were in the ranges 17-22 and 30-37 kcal/mol, respectively. At low coverage, the PEG monomers seemed firmly anchored to the protein surface and were not displaced by water, while only about 20% of MAA was replaced by water. The synthesis of MIPs was successfully with a monomer conversion higher than 99% and the production of spherical particles with average diameter of 344 ± 33 nm. HPLC analysis showed a specific recognition factor of MMP9 on MIPs of about 1.3. FT-IR Chemical Imaging confirmed the mechanisms necessary to generate a "selective memory" of the MIPs towards the enzyme. HPLC results indicated that the rebound amount of both TIMP1 and MMP2 to MIPs is lower than that of the template, showing a selectivity factor of 2.1 and 2.3, respectively. Preliminary tests on the effect of MIPs on H9C2 cells revealed that this treatment has no cytotoxic effects.