In the field of medicine, nanoparticles (NPs) have been used to improve imaging techniques in very early stages of the disease, to access different sites (e.g., crossing the blood-brain barrier) and deliver different therapeutic agents to target cells (e.g., targeting only cancer cells without interacting with the normal tissue) [1]. Furthermore, nanomaterials are employed extensively in the field of regenerative medicine to replace damaged tissue [2]. In cardiovascular diseases (CVDs), nanotechnologies have four main applications: diagnostics, molecular imaging, targeted drug delivery, tissue regeneration and engineering. This is due to the fact that nanomolecules act as carriers facilitating controlled release of imaging and diagnostic agents at the site of the cardiac injury [3].

CVDs are one of the most important global epidemics of the 21st century, with an estimated prevalence of more than 37 million patients globally. Furthermore, CVDs have a worldwide distribution with high mortality and hospitalization rates being associated with a poor quality of life for the patients and high costs for healthcare systems ^[4][5][4,5]. Out of the plethora of conditions that can lead to CVDs, atherosclerosis and ischemic heart disease are responsible for more than 2/3 of the cases progressing to severe heart failure (HF) and finally death. The continuous increase of ischemic HF incidence in the last century can probably be attributed to the aging population, the increase of sedentarism, poor nutrition and smoking. Accumulating evidence from cohort studies and randomized clinical trials (RCTs) has brought novel insight regarding prevention and treatment for HF, and the absolute survival rate in the past 30 years has increased 9%. Furthering our understanding of this condition with its molecular and genetic pathways can make this percentage even higher [6]. The main physiopathological feature of ischemic heart failure is ventricular remodeling, and it consists of myocyte hypertrophy with loss of myofibrils and abnormal intracellular matrix, myocardial fibrosis with excessive accumulation of extracellular matrix components, and changes in the collagen fibers proportions, inflammation and activation of the inflammatory pathways, mitochondrial disfunction and finally apoptosis and autophagy [7].

Current trends of HF pharmacological treatment focus on the adrenergic and renin–angiotensin–aldosterone systems (RAAS), which are responsible for the regulation of vasoconstriction and fluid retention. Agents that induce RAAS inhibition (ACE inhibitors, angiotensin 2 receptor blockers and aldosterone antagonists) were proven in multiple RCTs to promote cardiac remodeling and reduce HF symptomatology, morbidity and mortality. Activation of the sympathetic nervous system secondary to reduced cardiac output leads to increase total peripheral resistance, activation of beta-adrenergic receptors increasing wall tension, oxygen consumption and myocyte necrosis [5]. Other peptides such as natriuretic peptides, bradykinin, substance P and adrenomedullin promote diuresis, natriuresis, vasodilation and inhibit the adrenergic system. Attempts to administer exogenous natriuretic peptides have not shown any effectiveness in HF therapy, especially due to their increased degradation. However, inhibiting neprilysin (a catalyzer of natriuretic peptide degradation) was proven to decrease HF hospitalization rates and mortality ^[4][8][4,8]. Another target for HF therapy is represented by the SGLT2 inhibition, which regulates renal sodium absorption, renin secretion, a catabolic state with lower insulin and higher glucagon levels, and has favorable effects on myocardial metabolism ^[8][9][8,9].

In contrast, modern therapeutical approaches try to focus on promoting cardiac regeneration through different mechanisms such as: reprogramming of cardiac fibroblasts into contractile cells, cardiomyocyte proliferation stimulation, gene and cell-based therapies [10]. The main challenges of these therapies arise from the specific pathophysiology of HF and result from the ability of the delivering agent to identify, target and deliver molecules or cells at the level of the damaged myocardium. Furthermore, in cases of cell therapy it is vital to also facilitate retention and engraftment at the targeted area, together with cell monitoring over the course of treatment. Most of these challenges could be surpassed by using nanoparticles as delivery systems.

2. Nanotechnologies and Diagnostic of Ischemic Heart Disease

One of the main staples of ischemic heart disease and HF successful management is early detection, which enables administration of proper treatment, improving the prognostic, quality of life and survival of patients. Current means of diagnosis are based on a cumulus of clinical signs, assessment of different heart-specific biomarkers and imaging modalities. However, the onset of clinical signs and pathological imaging findings manifest later in the evolution of the disease, while detection of biomarkers in the human plasma and conventional imaging techniques have limited sensitivity and specificity. In this context, nanotechnologies have been employed in order to enhance the detection and description of physiopathological mechanisms of cardiovascular diseases. These mechanisms are atherosclerosis, thrombus formation and localization, myocardial infarction (MI), and postinfarction remodeling, angiogenesis and myocyte apoptosis [11]. Atherosclerosis is responsible for the development of heart failure directly in the context of ischemic heart disease and indirectly through hypertension, peripheral vascular disease and through promoting other comorbidities such as stroke and renal injury. Unstable atherosclerotic plaques are responsible for the majority of cases of myocardial infarction and sudden cardiac death. Current imaging techniques such as intravascular optical coherence tomography (OCT) can detect vulnerable plaques based on histopathological structure [12]. However, in order to better evaluate the severity of atherosclerotic plaques, a molecular analysis is necessary. Concentration of macrophages and their activity at the level of atherosclerotic plaque were shown to be an attractive indicator for plaque instability [13]. In this regard, nanoprobes composed of dextrinated and diethylenetriamine pentaacetate (DTPA) modified magnetofluorescent 20 nm nanoparticles have been used to target macrophages expressing CD68 at the level of atherosclerotic plaques. After coating this nanoprobe with ⁶⁴Cu, the accumulation of the probe at the level of atherosclerotic arteries of model apoE^−/− mice was demonstrated through PET/CT and in vivo MRI [14]. In another study, fluorescent magnetic nanoparticles conjugates were used to target vascular adhesion molecule-1 (VCAM-1) expressed by unstable plaques, and the accumulation of the nanoparticles was confirmed by MRI [15]. There are also studies that use intravascular photoacoustic (IVPA) imaging based on administration of silica-coated gold nanorods (SiO2AuNR), which are thermally stable nanosensors, thus enabling temperature mapping of the plaque’s activity and molecular composition. IVPA can be used in conjunction with intravascular ultrasound (IVUS) to evaluate both the morphology and the functionality of the plaque [16]. More information about pathological mechanisms involving atherosclerosis and NPs targeted therapies will be described later in this paper. Thrombosis is usually a consequence of plaque rupture and is the underlying cause of clinical manifestations such as myocardial infarction or stroke. Furthermore, thrombus formation is encountered in other cardiovascular pathologies such as atrial fibrillation, ventricular aneurysm and venous embolism (deep vein thrombosis and pulmonary embolism). In order to identify thrombi, researchers tried to use different molecular targets such as fibrin, cell-adhesion proteins and activated platelets. For example, the pentapeptide Cys-Arg-Glu-Lys-Ala has a high specificity for fibrinogen/fibrin complexes and when conjugated with a dye-conjugated H₂O₂-scavenging polymer it targets newly formed thrombi [17]. Other studies have focused on P-selectin, a cell-adhesion protein expressed on activated platelets, which can be targeted by gadolinium-targeted paramagnetic or iron oxide nanoparticles, which enhance the MR imaging of microthrombi ^[18][19][18,19]. In addition to identifying the location of the thrombus, MRI together with florescence imaging can be used to further evaluate the severity of myocardial infarction. MRI is an ideal imaging technique used for the identification of tissue scaring and myocardial contraction, while fluorescence imaging can assess the biological processes at the level of the ischemic myocytes. Magneto-fluorescent nanoprobes such as cross-linked iron oxide (CLIO)-Cy5.5 are accumulated in the macrophages at the level of the myocardial infarction [20]. Using them as an MRI enhancement agent may lead to further understanding of the role of cardiac macrophage mediated inflammation, encountered in postinfarction cardiac remodeling. CLIO-Cy5.5 can also be conjugated with annexin V in order to detect and possibly stabilize early apoptotic processes in ischemic heart diseases ^[21][22][21,22]. In fact, most of the aforementioned nanomolecules can be also used to act as medication carriers to the targeted cells. Nanotheranostics (therapy and diagnostics) is characterized by 3 stages. Stage 1 implies the nanoparticle-based diagnosis of the disease and the evaluation of conventional treatment efficiency. In stage 2, nanomolecules are used for therapy, and in stage 3, nanoparticle-based imaging is used to evaluate the efficacy of nanotherapy [23]. Nanoparticles used in the diagnosis and imaging of cardiovascular diseases are summarized in Table 1.

Nanoparticle	Molecular Mechanism	Imaging Technique
Dextrinated and DTPA modified magnetofluorescent [14]	target macrophages expressing CD68 at the level of atherosclerotic plaques	PET/CT and MRI of unstable atherosclerotic plaques
CLIO-Cy5.5 fluorescent magnetic nanoparticles conjugated with VHSPNKK polypeptide [15]	target vascular adhesion molecule-1 (VCAM-1) expressed by unstable plaques	MRI of unstable atherosclerotic plaques
CLIO-Cy5.5 conjugated with annexin V [21]	detecting apoptotic heart cells	MRI in ischemic heart disease and cardiomyopathies
Paramagnetic or iron oxide nanoparticles	P-selectin on activated platelets	MRI detection of thrombosis
AuNPs coated with collagen binding adhesion protein 35 [24]	myocardial scar tissue	alternative to iodinated contrast in CT imaging
		differentiation of stem cells into myocytes; cardiac regeneration and improved cardiac function	Ferumoxytol—paramagnetic iron oxide nanoparticle [25]	uptake at the level of activated macrophages in the ischemic myocardium	MRI in ischemic heart disease
Silica-coated gold nanorods (SiO2AuNR) [16]	detection of activated macrophages in unstable atherosclerotic plaques	intravascular photoacoustic (IVPA) temperature mapping of the plaque’s activity

3. Nanotechnologies and Therapy of Ischemic Heart Disease

Nanoparticles represent an ideal drug and molecule carrier as they are designed to elude the host’s immune system, have an acceptable biodegradability, biocompatibility, and have the ability to target a specific site. Furthermore, due to the size of these molecules they can pass through the cell membrane and target desired cellular components. Some of the most studied nanoparticles in cardiovascular diseases are liposomes, dendrimers, micelles, polymeric and inorganic nanocarriers [26]. The treatment of ischemic heart disease consists of two main constituents. The first one is the treatment of the occluded vessels, while the second one consists of facilitating cardiomyocyte survival and regeneration, inhibition of inflammation and macrophage activation. As mentioned before, nanoparticles can be used to target coagulation molecules or platelet adhesion in newly formed thrombi responsible for myocardial infarction. For example, a controlled release and thrombolytic activity of tissue-type plasminogen activator (tPA) and successful recanalization in swine MI models was attained through nanoparticles containing (tPA) coupled to von Willebrand factor [27]. Similarly, magnetic nanoparticles conjugated with urokinasis were directed for thrombolysis by a magnetic field at the site of the thrombus in an experimental mouse model [28]. In patients receiving a stent after an ischemic event, the risk of stent restenosis remains an important factor for the efficiency of the treatment. Nanocarriers that facilitate the retention of antiproliferative drugs at the level of the lesion of the endothelial tissue have been proposed as a prophylactic measure for stent restenosis. Lipid-based nanoparticles containing different classes of drugs such as bisphosphonate or prednisolone have been shown to reduce neointimal growth in atherosclerotic experimental models implanted with bare metal stents. Other polymeric-based nanoparticles such as polylactic-coglycolic acid (PLGA) nanoparticles were loaded with paclitaxel or sirolimus similar to drug eluting stent with acceptable results [29]. In 2007, the results of the systemic nanoparticle paclitaxel (nab-paclitaxel) for in-stent restenosis I (SNAPISTI) trial were reported, revealing that nab-paclitaxel doses under 70 mg/m² may be used to prevent stent restenosis [30]. Drug-eluting stents (DES) have had good results in preventing stent restenosis when compared to bare metal stents. However, DES inhibit the proliferation of the endothelial layer of the vessels, leading to an increased risk of thrombosis requiring long-term antithrombotic therapy. As a response to these issues, experimental studies have proposed various techniques to generate nanopatterns that promote proper endothelization on the surface of titanium stents [31]. In addition, nanoparticle-eluting stents have been produced using nanomolecules, promoting endothelization with a satisfactory internalization into the smooth vascular muscle cells [32]. During ischemia, the tissue enters an acidotic state that results in releasing intracellular content and production of reactive oxygen species (ROS). H₂O₂ is the most abundant form of ROS, and its production is associated with proinflammatory cytokine release, myocytes apoptosis and the development of ischemic heart disease [33]. To reduce the effects of H₂O₂, nanoparticles linked by peroxalate bonds and containing antioxidant molecules can be delivered to the ischemic area. The peroxalate bonds are cleaved by excess H₂O₂ and thus the antioxidant molecules are released at the site of the oxidative stress [33]. Superoxide dismutase 1 (SOD1, Cu/Zn SOD) is an enzyme responsible for the degradation of ROS, and its overexpression has been shown to have a protective effect in experimental models of myocardial infarction [34]. Polyketal particles were designed to carry SOD1 and the use of these nanoparticles has been proven to improve cardiac function in myocardial infarction mice sustained by echocardiographic measurements [34]. Inorganic nanoparticles are composed of an inorganic core surrounded by an organic/inorganic shell being designed to avoid the host’s immune system and thus increase biocompatibility. They are very well suited for theranostic purposes as the inorganic component can be used to enhance imaging techniques such as CT and MRI. Gold nanoparticles (AuNPs) are widely used because they are easily synthetized, they present low toxicity and immunogenicity, and have good stability. Due to these beneficial properties, AuNPs have been conjugated with drugs that are already used in clinical practice, such as levosimendan and beta-blockers. In an animal model of doxorubicin-induced HF, these aggregates showed significant cardio-protective effects [35]. Au NPs that accumulate in the ischemic heart tissue have been used to deliver and influence exogenous growth factors resulting in a 1.7-fold increase of blood perfusion at that level [23]. In the past decade, increasing evidence has shown that microRNAs (endogenous, conserved, single-stranded, non-coding RNAs of 21–25 nucleotides in length) have an important role in angiogenesis, apoptosis, cell growth, differentiation, cardiac cell contractility, control of lipid metabolism and plaque formation [36]. Among their multiple influences on cardiovascular and metabolism homeostasis, many experimental studies have shown that microRNAs serve as significant regulators and fine tuners of a variety of pathophysiological cellular effects and molecular signaling pathways involved in development of atherosclerosis [37]. AuNPs can be coupled with gene-regulatory molecules such as microRNA 155, increasing the expression of anti-inflammatory type 2 macrophages that mitigate local inflammation and myocytes apoptosis [38]. Albumin-polyvinyl alcohol-AuNPs nanofibrous scaffolds have also been shown to facilitate cardiogenic differentiation of mesenchymal stem cells ^[35][39][35,39]. Finally, AuNPs coated with collagen binding adhesion protein 35 can also be used as an alternative to iodinate contrast for the CT imaging detection of myocardial infarction [24]. Other inorganic NPs with excellent theranostic properties are iron oxide NPs (SPION). Due to their superparamagnetic properties, biodegradability and surface characteristics, they can be used as a contrast agent for MRI of myocardial infarction and myocarditis experimental models [40]. Furthermore, there are clinical phase III trials using ferumoxytol for detailed characterization of infarct pathology by causing hypoenhancement (in T2-weighted images) and signal void (in T2*-mapping images) [25]. SPIONs also present cardioprotective effects, as Fe₂O₃ NPs coated with dimercaptosuccinic acid decreased ROS production and increased nitric oxide secretion [41]. Another interesting approach was administration of inhalable biodegradable inorganic nanoparticles that can target myocardial cells. Researchers delivered negatively charged calcium phosphate NPs (CaPNPs), which accumulated into the myocardium 60 min after inhalation. After reaching the intracellular compartment of the myocytes, CaPNPs released a mimetic peptide (R7W-MP) that improved cardiac contractility by targeting the Cavβ2 cytosolic subunit of the L-type calcium channel (LTCC) [42]. Polymeric NPs are one of the largest and most versatile classes of NPs. Their main feature consists of their ability to be designed and tuned in order to accommodate a wide range of molecules (drugs, proteins, nucleic acids) and facilitate a proper release inside the cells. This class of NPs consists of amphiphilic micelles, vesicles, dendrimers and polymerosomes, all of which can be synthetized according to the configuration molecule they are supposed to deliver [43]. These properties are essential in the context of increasing research regarding gene regulation therapies through non-coding RNA and even gene delivery therapies. Out of non-coding RNAs, microRNAs (approx. 23 nucleotides) play important gene-regulatory roles by influencing post-transcriptional repression of coding messenger RNAs [44]. Heart-specific microRNAs have significant roles in cell function, cardiac regeneration and differentiation [10]. Combining polymeric NP composed of poly(9,9-dioctylfluorene-alt-benzothiadiazole) (PBFT) and 1,2-distearoylphosphatidyl-ethanolamine-PEG-amino (DSPEPEG-NH2) is an appealing approach to generate a polymeric matrix for the protection of miR-199a against enzymatic degradation. Experimental studies using an in vivo ischemic heart disease model showed that this miRNA-polymeric nanoparticle combination (miNP) promotes proliferation of endothelial stem-cell-derived cardiomyocytes in the detriment of cardiac fibroblasts, leading to cardiac muscle regeneration and scar reduction [45]. In another study, a pegylated dendrigraft poly-L-lysine (PEG-DGL) dendrimer coupled with an antisense oligonucleotide was used for the inhibition of miR-1. Administration of this miNP succeeded in reducing the extent of the myocardial infarction in mice models, most likely due to the inhibition of miR-1, which has an important role in signaling apoptosis [46]. MicroRNAs also play an important role in regulation of macrophage inflammatory response at the level of the ischemic cardiac tissue. Coupled miR-21, Ca²⁺ and hyaluronan-sulfate NPs (HASCa²⁺-miRNA) NPs were able to signal a shift of macrophage phenotype from proinflammatory to regenerative state [47]. Dendrimer polymers are also excellent carriers for larger polinucleotidic molecules genes such as small interfering RNAs, messenger RNAs and DNA for the targeted treatment of altered cardiac tissue ^[48][49][48,49]. In an experimental study, a polyethylene-glycol-modified polyamidoamine was loaded with the recombinant plasmid, arginine–glycine–aspartic acid peptide, and hirudine. This novel NP was efficiently used for thrombosis treatment in MI [23]. Finally, polymeric nanoparticles can also be used as promoters and mediators for cell-based therapies. For example, simvastatin-conjugated PLGA NPs can be coupled in vitro with adipose-derived stem cells. This new nanocomplex improved cardiac function as a result of endogenous cardiac-cell regeneration when administered to an MI animal model. The regeneration process was attributed to the constant release of simvastatin from the stem-cell-nanoparticles complex, rather than the direct effect of the stem cells. Furthermore, the number of cells required to deliver this NPs therapy was rather small (10.000 cells/mouse), thus decreasing other risks of cell administration such as thrombosis [50]. Nanoparticles developed for promoting cardiac regeneration are summarized in Table 2.