Cardiovascular diseases (CVDs) remain the leading cause of mortality worldwide, highliting the urgent need for new therapeutic strategies. Peptide-based therapies have demonstrated significant potential for treating CVDs; however, their clinical application is hindered by their limited stability in physiological fluids. To overcome this challenge, an effective drug delivery system is essential to protect and efficiently transport peptides to their intended targets. This study introduces two distinct strategies for loading a cardio-specific mimetic peptide (MP), previously designed to modulate L-type calcium channel function in cardiomyocytes, onto calcium phosphate nanoparticles (CaP NPs). MP-loaded CaP NPs were prepared by two different wet precipitation syntheses, one of which involved the use of sodium polyacrylate as a templating agent. Characterization of MP-loaded CaP NPs showed that their crystallinity, size, surface charge, and morphology could be tuned by adjusting the synthesis parameters. In vitro tests on cardiac cells confirmed that both types of MP-loaded CaP NPs are biocompatible with HL-1 cardiomyocytes and restored intracellular calcium flux under stressed conditions, highlighting their therapeutic potential. These results pave the way for further optimization of CaP NP formulations and suggest their potential as a viable nanomaterial for CVD treatment.

In the dynamic landscape of biomedical research, therapeutic RNA aptamers have recently come to the forefront, showing significant potential in diagnostics and therapeutics. This review aims to raise awareness of aptamer technology within the scientific community by exploring the progress made in the therapeutic field, from the lessons learned in research to the future opportunities and impact that these innovative molecules are increasingly having on society to meet current health needs, i.e. targeted and personalized therapies.

Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns—particularly those specific to this class of drugs—is crucial. Here, we propose an in vitro approach to screen for potential adverse off-target effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NA-drugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticle-based delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.

Regulation of mitochondrial Ca2+ uptake is critical in cardiac adaptation to chronic stressors. Abnormalities in Ca2+ handling, including mitochondrial uptake mechanisms, have been implicated in pathological heart hypertrophy. Enhancing mitochondrial Ca2+ uniporter (MCU) expression has been suggested to interfere with maladaptive development of heart failure. Here, we addressed whether MCU modulation affects the cardiac response to pressure overload. MCU content was quantified in human and murine hearts at different phases of myocardial hypertrophy. Cardiac function/structure were analyzed after Transverse Aortic Constriction (TAC) in mice undergone viral-assisted overexpression or downregulation of MCU. In vitro and ex vivo assays determined the effect of MCU modulation on mitochondrial Ca2+ uptake, cellular phenotype and hypertrophic signaling. In human and murine hearts MCU levels increased in the adaptive phase of myocardial hypertrophy and declined in the failing stage. Consistently, modulation of MCU had a cell-autonomous effect in cardiomyocyte/heart adaptation to chronic overload. Indeed, upon TAC MCU-downregulation accelerated development of contractile dysfunction, interstitial fibrosis and heart failure. Conversely, MCU-overexpression prolonged the adaptive phase of hypertrophic response, as, in advanced stages upon TAC, hearts showed preserved contractility, absence of fibrosis and intact vascularization. In vitro and ex vivo analyses indicated that enhancement in mitochondrial Ca2+ uptake in cardiomyocytes entails "mitochondrion-to-cytoplasm" signals leading to ROS-mediated activation of Akt, which may explain the protective effects towards heart response to TAC. Enhanced mitochondrial Ca2+ uptake affects the compensatory response to pressure overload via retrograde mitochondrial-Ca2+/ROS/Akt signaling, thus uncovering a potentially targetable mechanism against maladaptive myocardial hypertrophy.

Recombinant proteins, cell, and gene therapies are collectively defined as biological drugs or biologics. These therapies have transformed the lives of millions of patients over the past decades, with the number of FDA-approved biologics increasing exponentially in recent years. However, out of approximately 700 biological therapies approved by the FDA in the last 20 years, less than 1

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.

Summary: 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.

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.

•Aptamers, like monoclonal antibodies, have a unique structure that allows them to bind to their targets with high specificity and selectivity.•Aptamers are small and flexible synthetic molecules with little steric hindrance and scarcely immunogenic.•Aptamer-based drugs encompass a diverse range of technologies, including novel shapes, chemistries, and delivery methods, as well as an assorted use for therapeutic and diagnostic purposes.•Aptamers have the potential to complement and improve the management of CVDs.

Antibiotic resistance is a serious health and social problem that will have a substantial impact in the coming years on the world health and economy. Thus, the increasing demand for innovative antibiotics, has prompted many researchers in the medical, microbiological, and biochemical fields to exploit the properties of antimicrobial peptides (AMPs). When properly used, designed, and conveyed, AMPs can really represent a valid alternative to conventional drugs especially in situations that are particularly difficult to treat such as chronic infections found in Cystic Fibrosis (CF) patients. In this review we focused on the applications of AMPs in the specific field of CF, illustrating different types of peptides from natural, naturally modified, synthetic as well as the different strategies used to overcome the barriers, and the physiological conditions in which AMPs must operate.

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 mu g 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.

A dry powder for inhalation, composed of mannitol microparticles carrying calcium phosphate nanoparticles, has been developed for cardiac delivery of loaded bioactive peptide. The goal was to achieve highly efficient deposition of microparticles in the deep lung for release and translocation of drug loaded nanoparticles into blood, enabling a direct transport to the heart via the pulmonary vein. In this work, the relevant quality attributes of inhalable microparticles embedding nanoparticles were discussed. Furthermore, peptide drug release from microparticles was studied in vitro and correlated to microparticle dissolution and nanoparticle degradation.

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.

Aim: Sepsis-induced cardiomyopathy is commonplace and carries an increased risk of death. Melusin, a cardiac muscle-specific chaperone, exerts cardioprotective function under varied stressful conditions through activation of the AKT pathway. The objective of this study was to determine the role of melusin in the pathogenesis of lipopolysaccharide (LPS)-induced cardiac dysfunction and to explore its signaling pathway for the identification of putative therapeutic targets. Methods and results: Prospective, randomized, controlled experimental study in a research laboratory. Melusin overexpressing (MelOV) and wild-type (MelWT) mice were used. MelOV and MelWT mice were injected intraperitoneally with LPS. Cardiac function was assessed using trans-thoracic echocardiography. Myocardial expression of L-type calcium channel (LTCC), phospho-Akt and phospho-Gsk3-b were also measured. In separate experiments, wild-type mice were treated post-LPS challenge with the allosteric Akt inhibitor Arq092 and a mimetic peptide (R7W-MP) targeting the LTCC. The impact of these therapies on protein-protein interactions, cardiac function, and survival was assessed. MelOV mice had limited derangement in cardiac function after LPS challenge. Protection was associated with higher Akt and Gsk3-b phosphorylation and restored LTCC density. Pharmacological inhibition of Akt activity reversed melusin-dependent cardiac protection. Treatment with R7W-MP preserved cardiac function in wild-type mice after LPS challenge and significantly improved survival. Conclusions: This study identifies AKT/Melusin as a key pathway for preserving cardiac function following LPS challenge. The cell-permeable mimetic peptide (R7W-MP) represents a putative therapeutic for sepsis-induced cardiomyopathy.

Myocardial infarction causes 7.3 million deaths worldwide, mostly for fibrillation that electrically originates from the damaged areas of the left ventricle. Conventional cardiac bypass graft and percutaneous coronary interventions allow reperfusion of the downstream tissue but do not counteract the bioelectrical alteration originated from the infarct area. Genetic, cellular, and tissue engineering therapies are promising avenues but require days/months for permitting proper functional tissue regeneration. Here we engineered biocompatible silicon carbide semiconductive nanowires that synthetically couple, via membrane nanobridge formations, isolated beating cardiomyocytes over distance, restoring physiological cell-cell conductance, thereby permitting the synchronization of bioelectrical activity in otherwise uncoupled cells. Local in-situ multiple injections of nanowires in the left ventricular infarcted regions allow rapid reinstatement of impulse propagation across damaged areas and recover electrogram parameters and conduction velocity. Here we propose this nanomedical intervention as a strategy for reducing ventricular arrhythmia after acute myocardial infarction.

Calcium phosphate is a natural biomineral and the major inorganic constituent of bones and teeth. Therefore, synthetic calcium phosphates that mimic the biogenic ones possess excellent biocompatibility as well as biodegradability and are promising materials for medicine. Due to their unique physiochemical properties, calcium phosphate nanoparticles (CaP NPs) are extensively exploited in nanomedicine as carriers of biomolecules, including peptides, proteins, and nucleic acids. In this regard, peptides are of particular interest as they are exceptionally selective and efficacious for the treatment of a broad range of diseases. Among various peptides for biomedical applications, cardio-specific peptides are particularly interesting since they represent a valuable alternative to conventional treatments. Moreover, they can contribute to overcome important clinical limitations, including drug resistance and non-specific biodistribution of traditional drug products. In this work, we have investigated the loading of a therapeutic mimetic peptide, which was previously shown to improve myocardial contraction and results in the restoration of cardiac function. Peptide-loaded CaP NPs were prepared by exploiting a biomineralization approach, by using a mineralizing solution containing Ca2+, Mg2+, and PO43- ions. Several experimental conditions were tested by varying the reaction time, as well as the drug concentration. Colloidal stability, morphology, size, as well as drug loading were evaluated to identify the best candidate to be tested in vitro in the future.

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.

Inhalation of Calcium Phosphate nanoparticles (CaPs) has recently unmasked the potential of this nanomedicine for a respiratory lung-to-heart drug delivery targeting the myocardial cells. In this work, we investigated the development of a novel highly respirable dry powder embedding crystalline CaPs. Mannitol was selected as water soluble matrix excipient for constructing respirable dry microparticles by spray drying technique. A Quality by Design approach was applied for understanding the effect of the feed composition and spraying feed rate on typical quality attributes of inhalation powders. The in vitro aerodynamic behaviour of powders was evaluated using a medium resistance device. The inner structure and morphology of generated microparticles were also studied. The 1:4 ratio of CaPs/mannitol led to the generation of hollow microparticles, with the best aerodynamic performance. After microparticle dissolution, the released nanoparticles kept their original size.