Mitochondrial a Kinase Anchor Proteins in Cardiovascular Health and Disease: A Review Article on Behalf of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology.

Second messenger cyclic adenosine monophosphate (cAMP) has been found to regulate multiple mitochondrial functions, including respiration, dynamics, reactive oxygen species production, cell survival and death through the activation of cAMP-dependent protein kinase A (PKA) and other effectors. Several members of the large family of A kinase anchor proteins (AKAPs) have been previously shown to locally amplify cAMP/PKA signaling to mitochondria, promoting the assembly of signalosomes, regulating multiple cardiac functions under both physiological and pathological conditions. In this review, we will discuss roles and regulation of major mitochondria-targeted AKAPs, along with opportunities and challenges to modulate their functions for translational purposes in the cardiovascular system.

Optimization of In Vivo Studies by Combining Planar Dynamic and Tomographic Imaging: Workflow Evaluation on a Superparamagnetic Nanoparticles System.

Molecular imaging holds great promise in the noninvasive monitoring of several diseases with nanoparticles (NPs) being considered an efficient imaging tool for cancer, central nervous system, and heart- or bone-related diseases and for disorders of the mononuclear phagocytic system (MPS). In the present study, we used an iron-based nanoformulation, already established as an MRI/SPECT probe, as well as to load different biomolecules, to investigate its potential for nuclear planar and tomographic imaging of several target tissues following its distribution via different administration routes. Iron-doped hydroxyapatite NPs (FeHA) were radiolabeled with the single photon γ-emitting imaging agent [99mTc]TcMDP. Administration of the radioactive NPs was performed via the following four delivery methods: (1) standard intravenous (iv) tail vein, (2) iv retro-orbital injection, (3) intratracheal (it) instillation, and (4) intrarectal installation (pr). Real-time, live, fast dynamic screening studies were performed on a dedicated bench top, mouse-sized, planar SPECT system from t = 0 to 1 hour postinjection (p.i.), and consequently, tomographic SPECT/CT imaging was performed, for up to 24 hours p.i. The administration routes that have been studied provide a wide range of possible target tissues, for various diseases. Studies can be optimized following this workflow, as it is possible to quickly assess more parameters in a small number of animals (injection route, dosage, and fasting conditions). Thus, such an imaging protocol combines the strengths of both dynamic planar and tomographic imaging, and by using iron-based NPs of high biocompatibility along with the appropriate administration route, a potential diagnostic or therapeutic effect could be attained.

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy.

The mitochondrial Ca2+ uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca2+ influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca2+ uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to ß-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca2+ uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of ß-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with ß-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy.

Novel Basic Science Insights to Improve the Management of Heart Failure: Review of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology.

Despite important advances in diagnosis and treatment, heart failure (HF) remains a syndrome with substantial morbidity and dismal prognosis. Although implementation and optimization of existing technologies and drugs may lead to better management of HF, new or alternative strategies are desirable. In this regard, basic science is expected to give fundamental inputs, by expanding the knowledge of the pathways underlying HF development and progression, identifying approaches that may improve HF detection and prognostic stratification, and finding novel treatments. Here, we discuss recent basic science insights that encompass major areas of translational research in HF and have high potential clinical impact.

MicroRNA-199a-3p and MicroRNA-199a-5p Take Part to a Redundant Network of Regulation of the NOS (NO Synthase)/NO Pathway in the Endothelium.

Objective- Members of the microRNA (miR)-199a family, namely miR-199a-5p and miR-199a-3p, have been recently identified as potential regulators of cardiac homeostasis. Also, upregulation of miR-199a expression in cardiomyocytes was reported to influence endothelial cells. Whether miR-199a is expressed by endothelial cells and, if so, whether it directly regulates endothelial function remains unknown. We investigate the implication of miR-199a products on endothelial function by focusing on the NOS (nitric oxide synthase)/NO pathway. Approach and Results- Bovine aortic endothelial cells were transfected with specific miRNA inhibitors (locked-nucleic acids), and potential molecular targets identified with prediction algorithms were evaluated by Western blot or immunofluorescence. Ex vivo experiments were performed with mice treated with antagomiRs targeting miR-199a-3p or -5p. Isolated vessels and blood were used for electron paramagnetic resonance or myograph experiments. eNOS (endothelial NO synthase) activity (through phosphorylations Ser1177/Thr495) is increased by miR-199a-3p/-5p inhibition through an upregulation of the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) and calcineurin pathways. SOD1 (superoxide dismutase 1) and PRDX1 (peroxiredoxin 1) upregulation was also observed in locked-nucleic acid-treated cells. Moreover, miR-199a-5p controls angiogenesis and VEGFA (vascular endothelial growth factor A) production and upregulation of NO-dependent relaxation were observed in vessels from antagomiR-treated mice. This was correlated with increased circulated hemoglobin-NO levels and decreased superoxide production. Angiotensin infusion for 2 weeks also revealed an upregulation of miR-199a-3p/-5p in vascular tissues. Conclusions- Our study reveals that miR-199a-3p and miR-199a-5p participate in a redundant network of regulation of the NOS/NO pathway in the endothelium. We highlighted that inhibition of miR-199a-3p and -5p independently increases NO bioavailability by promoting eNOS activity and reducing its degradation, thereby supporting VEGF-induced endothelial tubulogenesis and modulating vessel contractile tone.

APJ acts as a dual receptor in cardiac hypertrophy.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of ß-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

Peptidomimetic Targeting of Cavß2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

MicroRNA-133 modulates the ß1-adrenergic receptor transduction cascade.

RATIONALE: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate ß-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of ß-adrenergic receptors leads to impaired cardiac function, and ß-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. OBJECTIVE: To determine whether miR-133 affects ß-adrenergic receptor signaling during progression to heart failure. METHODS AND RESULTS: Based on bioinformatic analysis, ß1-adrenergic receptor (ß1AR) and other components of the ß1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective ß1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic ß1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. CONCLUSIONS: miR-133 controls multiple components of the ß1AR transduction cascade and is cardioprotective during heart failure.

MicroRNA-133 controls cardiac hypertrophy.

Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.

Diagnostic and Therapeutic Aptamers: A Promising Pathway to Improved Cardiovascular Disease Management.

Despite advances in care, cardiovascular diseases remain the leading cause of death worldwide. As a result, identifying suitable biomarkers for early diagnosis and improving therapeutic and diagnostic strategies is crucial. Because of their significant advantages over other therapeutic approaches, nucleic-based therapies, particularly aptamers, are gaining increased attention. Aptamers are innovative synthetic polymers or oligomers of single-stranded DNA (ssDNA) or RNA molecules that can form 3-dimensional structures and thus interact with their targets with high specificity and affinity. Furthermore, they outperform classical protein-based antibodies in terms of in vitro selection, production, ease of modification and conjugation, high stability, low immunogenicity, and suitability for nanoparticle functionalization for targeted drug delivery. This work aims to review the advances made in the aptamers' field in biomarker detection, diagnosis, imaging, and targeted therapy, which highlight their huge potential in the management of cardiovascular diseases.

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure.

BACKGROUND: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. OBJECTIVES: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. METHODS: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. RESULTS: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. CONCLUSIONS: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.

NF-κB mediated miR-26a regulation in cardiac fibrosis.

Micro-RNAs (miRNAs) are a class of small non-coding RNAs, recently emerged as a post-transcriptional regulator having a key role in various cardiac pathologies. Among them, cardiac fibrosis that occurs as a result from an imbalance of extracellular matrix proteins turnover and is a highly debilitating process that eventually lead to organ dysfunction. An emerging theme on is that miRNAs participate in feedback loop with transcription factors that regulate their transcription. NF-κB, a key transcription factor regulator controls a series of gene program in various cardiac diseases through positive and negative feedback mechanism. But, NF-κB mediated miRNA regulation in cardiac fibrosis remains obscure. Bioinformatics analysis revealed that miR-26a has targets collagen I and CTGF and possesses putative NF-κB binding element in its promoter region. Here, we show that inhibition of NF-κB in cardiac fibroblast restores miR-26a expression, attenuating collagen I, and CTGF gene expression in the presence of Ang II, conferring a feedback regulatory mechanism in cardiac fibrosis. The target genes for miR-26a were confirmed using 3'-UTR luciferase reporter assays for collagen I and CTGF genes. Using NF-κB reporter assays, we determine that miR-26a overexpression inhibits NF-κB activity. Finally, we show that miR-26a expression is restored along with the attenuation of collagen I and CTGF genes in cardiac specific IkBa triple mutant transgenic mice (preventing NF-κB activation) subjected to 4 weeks transverse aortic banding (TAC), compared to wild type (WT) mice. The data indicate a potential role of miR-26a in cardiac fibrosis and, offer novel therapeutic intervention.

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo.

RATIONALE: MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. OBJECTIVE: The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. METHODS AND RESULTS: We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. CONCLUSIONS: Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Molecular mechanisms underlying the beneficial effects of exercise and dietary interventions in the prevention of cardiometabolic diseases.

Cardiometabolic diseases still represent a major cause of mortality worldwide. In addition to pharmacological approaches, lifestyle interventions can also be adopted for the prevention of these morbid conditions. Lifestyle changes include exercise and dietary restriction protocols, such as calorie restriction and intermittent fasting, which were shown to delay cardiovascular ageing and elicit health-promoting effects in preclinical models of cardiometabolic diseases. Beneficial effects are mediated by the restoration of multiple molecular mechanisms in heart and vessels that are compromised by metabolic stress. Exercise and dietary restriction rescue mitochondrial dysfunction, oxidative stress and inflammation. They also improve autophagy. The result of these effects is a marked improvement of vascular and heart function. In this review, we provide a comprehensive overview of the molecular mechanisms involved in the beneficial effects of exercise and dietary restriction in models of diabetes and obesity. We also discuss clinical studies and gap in animal-to-human translation.

Inhalable Microparticles Embedding Biocompatible Magnetic Iron-Doped Hydroxyapatite Nanoparticles.

Recently, there has been increasing interest in developing biocompatible inhalable nanoparticle formulations, as they have enormous potential for treating and diagnosing lung disease. In this respect, here, we have studied superparamagnetic iron-doped calcium phosphate (in the form of hydroxyapatite) nanoparticles (FeCaP NPs) which were previously proved to be excellent materials for magnetic resonance imaging, drug delivery and hyperthermia-related applications. We have established that FeCaP NPs are not cytotoxic towards human lung alveolar epithelial type 1 (AT1) cells even at high doses, thus proving their safety for inhalation administration. Then, D-mannitol spray-dried microparticles embedding FeCaP NPs have been formulated, obtaining respirable dry powders. These microparticles were designed to achieve the best aerodynamic particle size distribution which is a critical condition for successful inhalation and deposition. The nanoparticle-in-microparticle approach resulted in the protection of FeCaP NPs, allowing their release upon microparticle dissolution, with dimensions and surface charge close to the original values. This work demonstrates the use of spray drying to provide an inhalable dry powder platform for the lung delivery of safe FeCaP NPs for magnetically driven applications.

MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension.

MicroRNAs play an important role in myocardial diseases. MiR-133a regulates cardiac hypertrophy, while miR-29b is involved in cardiac fibrosis. The aim of this study was to evaluate whether miR-133a and miR-29b play a role in myocardial fibrosis caused by Angiotensin II (Ang II)-dependent hypertension. Sprague-Dawley rats were treated for 4 weeks with Ang II (200 ng/kg/min) or Ang II + irbesartan (50 mg/kg/day in drinking water), or saline by osmotic minipumps. At the end of the experimental period, cardiac miR-133a and miR-29b expression was measured by real-time PCR, and myocardial fibrosis was evaluated by morphometric analysis. A computer-based prediction algorithm led to the identification of collagen 1a1 (Col1A1) as a putative target of miR-133a. A reporter plasmid bearing the 3'-untranslated regions (UTRs) of Col1A1 mRNA was constructed and luciferase assay was performed. MiR-133a suppressed the activity of luciferase when the reporter gene was linked to a 3'-UTR segment of Col1A1 (P < 0.01). Mutation of miR-133a binding sites in the 3'-UTR of Col1A1 mRNA abolished miR-133a-mediated repression of reporter gene activity, showing that Col1A1 is a real target of miR-133a. In vivo, Ang II caused an increase in systolic blood pressure (P < 0.0001, tail cuff) and myocardial fibrosis in presence of a decrease in miR-133a (P < 0.01) and miR-29b (P < 0.01), and an increase in Col1A1 expression (P < 0.01). These effects were abolished by Ang II administration + irbesartan. These data demonstrate a relationship between miR-133a and Col1A1, suggesting that myocardial fibrosis occurring in Ang II-dependent hypertension is regulated by the down-regulation of miR-133a and miR-29b through the modulation of Col1A1 expression.

Altered ß-adrenergic response in mice lacking myotonic dystrophy protein kinase.

The protein kinase product of the gene mutated in myotonic dystrophy 1 (DMPK) is reported to play a role in cardiac pathophysiology. To gain insight into the molecular mechanisms modulated by DMPK, we characterize the impact of DMPK ablation in the context of cardiac ß-adrenergic function. Our data demonstrate that DMPK knockout mice present altered ß-agonist-induced responses and suggest that this is due, at least in part, to a reduced density of ß(1)-adrenergic receptors in cardiac plasma membranes.

Biocompatible antimicrobial colistin loaded calcium phosphate nanoparticles for the counteraction of biofilm formation in cystic fibrosis related infections.

The use of inhalable nanoparticles (NPs) for cystic fibrosis (CF) has been advocated as a promising tool to improve the efficacy of antimicrobials taking advantage of their ability to penetrate airway mucus and pathogen biofilm and to release the drug in or in proximity to the enclosed bacteria. Here, inhalable calcium phosphate (CaP) NPs were functionalized with colistin (Col) which is one of the most active antimicrobials against Gram-negative bacteria. The adsorption kinetic and isotherm of Col on CaP-NPs were investigated and fitted according to different mathematical models and revealed an electrostatic interaction between positively charged amine groups of Col and negatively charged surface of CaP-NPs. The maximum Col payload was of about 50 mg g-1 of CaP-NPs. After functionalization, despite an increase of size (213 vs 95 nm), in citrate solution, CaP-NPs maintained a dimension and surface charge considered suitable for crossing mucus barrier. CaP-NPs do not interact with mucin and are able to permeate a layer of artificial mucus. In vitro tests on pulmonary cells demonstrated that CaP-NPs are not cytotoxic up to a concentration of 125 µg mL-1. The antimicrobial and antibiofilm activity of Col loaded CaP-NPs tested on Pseudomonas aeruginosa RP73, a clinical strain isolated from a CF patient, was similar to that of free Col demonstrating that the therapeutic effect of Col adsorbed on CaP-NPs was retained. This work represents the first attempt to use CaP-NPs as delivery system for the CF treatment. The encouraging results open the way to further studies.