Therapeutic applications of nanozymes and their role in cardiovascular disease

Enzymes use as a catalyst in biology that converts substrates into products in any biochemical reaction which needs a catalyst. The fi rst enzyme, urease, was said to be a protein in 1926, determined by James B. Since, enzymes well treated to be proteins that allow the enzymes to accomplish high activity as a catalyst. So, protein activity is missed when its exposure occurs at high temperature and high pH. Proteins are also affected by proteases present in the environment moreover natural enzymes have a high cost due to these practical applications of enzymes retard in industry [1].


Introduction
Enzymes use as a catalyst in biology that converts substrates into products in any biochemical reaction which needs a catalyst. The fi rst enzyme, urease, was said to be a protein in 1926, determined by James B. Since, enzymes well treated to be proteins that allow the enzymes to accomplish high activity as a catalyst. So, protein activity is missed when its exposure occurs at high temperature and high pH. Proteins are also affected by proteases present in the environment moreover natural enzymes have a high cost due to these practical applications of enzymes retard in industry [1].
However, enzyme mimetic is an increasing pull to overcome the limitations; enzyme mimicking is done with the help of nanozymes [2]. Nanozymes contain nonmaterial which have enzyme-like activity; this similar activity attracts the researchers. Nanozymes studies showed that they are extraordinary in many aspects, such as robustness to a harsh environment, long term storage, easy mass production, and high stability [3]. Nanozymes have different structures or sizes because nanomaterials are hard due to a porphyritic nucleus, and proteins are soft [4]. So the comparison with natural enzymes nanozymes is more stable because the nanomaterials they contain, are inorganic [2]. Nanozymes have inherent properties, and unique catalytic activities due to this nanozymes have great attention towards mimetic the natural enzymes until now 50 kinds of nanoparticles have been found. Catalytic activities of enzymes are Catalase (CAT), Peroxidase (POD), glucose oxidase [5], Superoxide Dismutase (SOD). Nanozymes are mainly divided into carbon-based, noble metal nanozymes, metal oxide nanozymes [1].
The many latest developments in the fi eld of nanotechnology have broadened the approach to investigate the novel enzymes. The possible catalytic mechanism for nanozymes has unravelled when combined with computer theoretical calculations, simulation. These studies improve the nanozymes catalytic activities. In biomedical technology, nanozymes have exhibited advantages in diagnosing and treating disease [7].

Antiinfl ammatory effects
Infl ammation plays a role in diabetes, physiology of arthritis, irritable bowel syndrome, heart disease, Alzheimer's disease, illnesses, and Parkinson's disease. The major constituent of the yellow spice turmeric, curcumin derived from the Curcuma longa. It is non-toxic and safe and has antiinfl ammatory properties [11]. A high level of nitric oxide [12] abnormally contributes to chronic infl ammation and immunological disorder resulting in organ damage. Cerium oxide nanoceria has characteristics used in nanotherapeutics and decreases the moderator chronic infl ammation [13]. Nanoceria can scavenge the reactive oxygen species [8] or free radicals and inhibit the mediators of infl ammation from protecting the cell against the infl ammatory diseases [4].
Catalase, glutathione peroxidase, and superoxide dismutase 1 (SOD1) are potent scavengers of ROS, in eye diseases, these enzymes are very effective because eye disease is related to oxidative stress. However, With the help of sodium hydroxide, acute corneal infl ammation in animals is treated. Eye disease was treated by contingent ordination of eye drops; these drops contain many problems, so due to this reveal that nanoparticles improve the effi cacy by drug carrier system or conquered the corneal diffusion barriers [14].

Antioxidant effects
ROS such as H 2 O 2 , O 2 • -, and •OH, are the by-products of metabolism. They can serve as second messengers at a low level and take part in many signalling processes [15]. Moreover, when the reactive oxide species are over-expressed, they lead to many unfavourable reactions; for example, over-expression of ROS produces oxidative damage to DNA, lipids, and proteins.
They also induce cell apoptosis and many other diseases like cancers, atherosclerosis, kidney diseases, arthritis, and neurodegeneration [16]. Antioxidants protect the tissues from damages compel by the free radicals; It also improves the many other damaged tissues. Antioxidants are catalase, glutathione reductase, glutathione peroxidase, peroxiredoxins, and superoxide dismutase. Biological antioxidant required some elements which have no antioxidant activity but needed for the proper functioning of biological antioxidant; these include iron, selenium, manganese, zinc, and copper( [17]. The V 2 O 5 has glutathione peroxidase (GPx) like antioxidant activity; it protects the cell from oxidative stress [18].
According to some new research, the Hollow PB nanozymes (HPBZs) contain multienzyme-like properties such as neuroprotection, and they scavenge the ROS( [19] ( Figure 1).
The over-production of ROS effect biomolecules leads to a wide range of human diseases, and induce tissue injury. Human skin which acts as a protective layer between the external environment and body is prone to pro-oxidant agents. ROS's over-production causes infl ammatory and allergic skin disease, for example, psoriasis, urticaria, and atopic dermatitis [20].  [21]. Due to their instability in biological buffers in vivo applications of PB nanoparticles are limited; the stability of these particles has been improved by coating with poly(vinylpyrrolidone), poly(diallyl dimethylammonium chloride), and polyethene glycol, from all these the chitosan, is best due to its excellent biocompatibility, good stabilization, biosafety, and biodegradability.
Hyperion Oh, et al. developed PB/Chi nanoparticles using many chitosan samples ranging from 3 to 100 kDa to improve the antioxidant activity and stability PB nanoparticles ( Figure 2).
Chitosan is a polysaccharide that is a permeation enhancer and mucoadhesive that facilitates NP retention in the administration [22]. The improvement in stability was examined by monitor change in the diameter of the nanoparticles and appearance.

Production cost
In vivo low cost of production due to usage of low dose.
The high cost of production and cause environmental pollution.
The high cost of production.
High cost to extract and in vivo usage.

Stability
Highly stability and low biotoxicity [42]. Instable in the biological environment [43]. Not stable. Practical applications of the natural agent are crucial due to low stability [44].

Neuroprotection
Stroke is the third headmost cause of death which is also called cerebral ischemia. Bioenergetics failure occurs due to the reduction of oxygen and glucose delivery to the brain during a stroke. These conditions lead to stress, blood-brain barriers, and ultimately cell death occurs. Cerium oxide nanoparticles have the potential to treat the stroke. Cerium is a metal that belongs to the lanthanide series of the periodic table. When oxygen is combined with the cerium in a formulation of nanoparticles, they have also found antioxidant properties [24]. Cell line HT22 is the most well stood clonal cell system for analyzing the oxidative stress. The natural pro-oxidant for HT22 cells is the glutamate amino acid when glutamate is present, cysteine consumption is blocked, and oxytocic occurs. Reduction of C 4 + to C 3 + and loss of oxygen is retained by creating an oxygen vacancy. This is done with the help of ceria nanoparticles, and due to these reactions, catalytic application possible [25].

Antithrombosis effect
Bacterial infection and thrombus formation are two major harms. These harms lead to complications in biomedical devices such as heart valves, catheters, and vascular grafts. To develop the antimicrobial and antithrombotic is important to sustain the function of these biomedical devices [26]. Colloidal Mesoporous Silica (CMS) nanoparticles have engrossed enormous consideration as possible injectable drug delivery.
In addition to their high Biodegradability and biocompatibility, CMS can selectively customize at their outer and inner surfaces to encapsulate molecules powerfully. They can be specifi cally functionalized on their outer surface [27]. The delivery of this multiple function CMS in the bloodstream prevents the thrombogenic effects. The coating of ethylene glycol addresses hemolytic behaviour and their fast degradation prevention.
Moreover, the additional efforts are critical to delivering these manifold core-shell CMS to the blood and assurance suffi cient circulation time to prevent the thrombotic effect. Heparin, anionic polysaccharide and highly sulfated, is best due to its anticoagulant property. Moreover, Heparin is conventionally injected intravenously or subcutaneously [28].
Heparin in free form involves an increased risk of bleeding due to these risks. Many other side effects occur. The example of this situation is this Heparin-induced thrombopenia even as diffusing all over the blood circulation system. Tissue targeted nanoceria the CMS with Heparin must be harmless and injectable into the bloodstream. Heparin and nanoceria prevent the clotting cascade's establishment only in the abrupt vicinity of nanocarriers and have no side effects [29].
Nitric oxide [12] is predictable as an antiplatelet agent. Nitric oxide [12] can prevent thrombus formation. Due to its ability to increase the cyclic Guanosine Monophosphate (cGMP) levels within the platelets, Nitric oxide [12] showed the antiplatelet agents. So, lowering the intracellular Ca 2+ level required for the common passageway in the coagulation cascade, NO drug agents' release to accomplish the antiplatelet activity in vivo.
These in vivo studies attract to mimic the natural role of NO release from the endothelial cells. Endothelial cells are healthy blood vessels [30].
NO was arising from the oxidation of L arginine and catalyzed by the enzyme nitric oxide synthase. Moreover, these enzymes use O 2 as an oxidant and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) as a cofactor. When the cofactor supplies and L arginine concentration are inadequate, the NO reduced form nitroxyl (HNO) produced [30]. Analogous to NO the HNO has an anti-thrombogenic effect. The NO generating catalysts and non-releasing materials have some problems, HNO is the possible solution.
However, there is no report on the local production of HNO effi ciently prevent thrombus formation. Some reports showed Graphene hemin glucose oxidase's conjugate use as a catalyst to generate HNO from L arginine and physiologically abundant glucose. These catalysts create a long-lasting antithrombotic covering [31].

Nanozymes role in cytoprotection
Platinum is used as an industrial catalyst in an extensive range. Some older studies about Platinum reported that the Pt NPs serve as both SOD mimics to scavenge H 2 O 2 , CAT, and O 2 • - [32]. Conventional methods also used poly (acrylic acid) (PAA)  https://www.peertechz.com/journals/international-journal-of-nanomaterials-nanotechnology-and-nanomedicine Citation: Nashat [32].
The non-cytotoxic properties of Pt NPs have been reported in previous research. All these studies showed that 10 -4 -10 3 ng/cm 2 (7.86×10 -7 -7.86 mg/l) of 20-100 nm Pt NPs affected neither the cellular metabolism nor cell death [33]. Moreover, hybrids nanomaterials have been broadly invented. Graphene oxide [33] nanomaterials broadly use in research, due to their high conductivity, absorbing properties, large surface area, and outstanding biocompatibility [34].
However, the hybrid GO-Se nanomaterials have GPx like antioxidant properties for Cytoprotection. In the presence of GSH, the GPx catalyzes the breakdown of H 2 O 2 to the harmless product. These nanomaterials catalyze the decomposition of H 2 O 2 to the less reactive product. Comparison between Se NPs and GO-Se nanoparticles showed higher GPx mimic catalytic effectiveness. Moreover, GO-Se nanozymes are obsessed with a high capacity to scavenging the reactive oxygen species [35].
Liu and his co-workers constructed enzyme from the selfassembly of polymers with protein; this nanocomposite is used for cytoprotection. Protein (SP1) is a stress-responsive protein and has no sequence homology to other stress proteins. SOD and GPx's catalytic centre assembled into protein (SP1) and polymer nanoparticles (PD5). The electrooptic contact between polymer and protein can self-assemble and form nanowires with SOD and GPx like properties (Figure 3). These nanocomposites scavenge the overexpressed reactive oxygen species and maintain the intracellular balance [36].

Anti-aging eff ect
Ageing is determined by the combination of environmental factors and genetics. Caenorhabditis elegans have identifi ed many genes such as daf-16, Age-1, Sir2, and daf-2. From which the daf-16, daf-2, Age-1 are associate with insulindependent signalling. Moreover, these genes are involved in oxidative stress responses and lead to stress resistance and longevity [37].
The redox reaction involves ageing; the detoxifi cation of ROS regulates ageing. The ageing-related diseases are preventing by the help of ROS scavenging enzymes. Quick, et al. [37]reported that C 60 [C(COOH) 2 ] 3 based SOD mimic showed fascinating anti-ageing effects and also better mice cognition ability [37]. Comprehensive studies showed that the nanozymes lower the age-related mitochondrial superoxide and improve the biological functions of mitochondria. Moreover, these biological functions rescued the age-related cognitive destruction due to its presence in mitochondria [38].
However, the drosophila melanogaster has been used to explain biological processes' mechanisms, including ageing, metabolism, and developments. Moreover, drosophila's short life cycle makes a suitable tool to study ageing mechanisms Results showed that the aged drosophila has a low climbing ability. The daily uptake of 200μg/mL Fe 3 O 4 NPs reduced the ROS level and improved climbing ability [38].

Anti-bacterial effect
Bacteria produce many contagious diseases that have major health issues all over the world. Millions of peoples died due to bacterial related issues; anti-bacterial agents are primarily antibiotics [40]. Moreover, metal-containing inorganic salts, organic agents, and natural anti-bacterial agents are used as anti-bacterial materials. These antimicrobial agents have drawbacks ( Table 2) [41]. So it is essential to induce stable, effi cient, and novel antimicrobial agents to overcome bacterial diseases. The peroxidase constituent of cytochrome P450, take part in many essential living functions.
Peroxidase catalyzes the H 2 O 2 to generate an elevated level of •OH and oxidize abundant organic substrates. The Qu and co-workers productively implement Graphene quantum dots

Dental biofi lms
Nanozymes also use to deal with oral diseases associated with biofi lms. Bacterial biofi lms bacteria that stay on the surface of contact, polysaccharides, secreting proteins, and biomolecules. Biomolecules enfold the bacteria due to these bacteria aggregates. As a result of these biofi lms, many human diseases are produced. The most costly and common biofi lms associated with the oral disease are dental cavities and nanozymes to deal with these oral diseases [45]. When bacteria, especially Streptococcus mutants, use the food sugar to produce the polysaccharides and focus them on the upper layer of the teeth, the environment will gradually be more acidic due to this cavities biofi lms formed because bacteria can be entrenched in the extracellular surface. Hence, it is hard to remove the oral biofi lm [46]. Moreover, the acidic environments in the biofi lm region can liquesce the analeptic component of teeth resulting in the commencement of dental cavities. Given the exclusive properties of enzymes, Koo's group used Fe 3 O 4 nanoparticles with CAT NPS as the peroxidase mimics to prevent oral bacterial biofi lm [2,47]. At the low pH environment, the oral biofi lm CAT NPs could catalyze the production of •OH to kill the embedded bacteria and mutilate the biofi lm matrix. CAT NPS alleviates the ossifi ed of apatite in acidic pH and protects the teeth ideally.
Moreover, the CAT NP/H 2 O 2 system keeps adequately hinder the formation and decadence of cavities, on mucosal tissue with no side effects. They also showed that ferumoxide exhibit properties of peroxidase in a pH reliant manner. These nanoparticles bind under the ultra-structure of biofi lm due to this bacterial membrane disturb. They also used rodent models of early childhood caries and ex vivo biofi lm; fi nding showed that acid damage of the enamel surface and contingent applied ferumoxide effectually suppresses biofi lm amassing, also prevents the start of tooth cavitation. Ferumoxide NPs serve as an effi cient therapy for biofi lm-related oral diseases [48].

Nanozymes role in cardiovascular disease
In the United States, over 80 million peoples have one in three deaths recognized for Cardiovascular Disease (CVD). Post myocardial infarction [1] heart failure can cause common deaths due to cardiovascular diseases. Mechanisms showed that in the place of damaged myocardium collagenous scar formation, these scars are non-contractile and lead to ventricular dilation, infarct wall thinning and hypertrophy of the myocardium. If no particular treatment applied and left untreated, this failure progress due to this heart no longer supplies adequate blood to the body, which is distinct as heart failure [49]. Moreover, advanced therapies of CVD and current techniques for early detection are imperfect, and they also have less effi ciency in preventing the diseases [8].
Nanotechnology involves the following constituents, human-made nature and the unique characteristics of new material and nanoscale dimensions of the whole system or its vital components. Cardiovascular nanomedicine aims to improve detection and address current CVD and therapy challenges by advancing in vivo and ex vivo biomarkers imaging and detection and improving tissue regeneration and drug delivery. Drugs are delivering with direct injection and nanocarriers [50].

Nanocarriers
Nanocarriers are nanomaterials with an affi nity to wrap

Liposomes
Liposomes size ranges from (80-300nm) they are lipid bilayer structure due to this use in medicine, and gene delivery.
Liposomes are lipophilic and can pass target tissues/cell membranes [52]. These spherical vesicles have been most extensively investigated in nanomedicine with reapproval due to small toxicity for in vivo experiments. They have superior biocompatibility because liposome consists of phospholipids to form bilayer inside aqueous phase [53]. To protect the agents from degradation, they can also incorporate hydrophobic agents inward the lipid bilayer and hydrophilic therapeutic agents inside the aqueous place. The liposome's advantages, such as high stability in biological environments, high agent-loading effi ciency, controllable release kinetics, the biodistribution of theragnostic agents, and biocompatibility provide liposome with effi cient pharmacokinetics [54]. By the process of encapsulation, the drug is fused in the liposomes. Moreover, liposomes are employed as coordinators to deliver particles under the skin layers due to this frequency of administration reduce [55].

Micelles
Micelles consist of amphiphilic artifi cial molecules. To form monolayer micelles self-gather in aqueous solution with a hydrophobic phase, they incorporate hydrophobic agents.
The micelles' size ranges from 10-100nm, and the inner space is more compassed than of liposomes [53]. These nanocarriers possessed of stimuli perceptive amphiphilic chunk copolymer are of pursuit due to their aptitude utilization in the fi eld of discipline drug delivery [56].

Polymeric nanoparticles
Nanomedicine defi nes as the device of therapeutics and/ or diagnostics on the nanoscale. Nanoscale gives benefi ts due to a huge degree of concurrent transfer and the prevention, treatment, and diagnosis of diseases in the biological systems [57]. Therapeutics based on polymeric nanoparticles are developed to advance the treatment and diagnosis of an extensive range of diseases. Leading in polymerization has implemented the engineering of leaded multifunctional polymeric NPs with accurate control, size, fi ctionalization, shape, and surface charge [58].
Advantage of these multifunctional polymeric NPs, where the rendition of multiple functional groups from nanoparticle manufacture empowers binding capabilities examined to linear polymers and higher cell recognition. Moreover, these polymers allow the encapsulation of cargo molecules that contain the drug and released at the targeted sites. In biosensing and photodynamic therapies fabrication of polymer, nanoparticles provide new avenues [58].
Poly(ethylene glycol) (PEG) is a hydrophilic, biocompatible, biologically inert polymer that has been permitted by the U.S. Food Drug Administration (FDA) for use in a wide range of application. The number and length of PEG cuffs per NPs have been shown to fabricate signifi cant deviation in circulation time in blood vs clearance of organs by eliminating polymeric NPs [57]. Flourishing data supports the idea that NPS loaded coated drugs onto an angioplasty balloon surface is an assuring approach to moderately enhance and protract drug delivery to the wall of arteries [59]. Nanoparticles are made from poly (lactic-co-glycolic acid) (PLGA) which is a biodegradable polymer that has been used as a vehicle of drug delivery for continuous releases of both hydrophilic and hydrophobic drugs [22].

Atherosclerosis
Atherosclerosis is an analytical progression that involves the gathering of cholesterol underneath the intima of arteries. These progressions are pursued by local commencement of the immune system, deposition of connective tissues, and vascular smooth muscle cells [63]. Moreover, atherosclerotic plaque proceeds from the early atheromatous lesion. When the atheromatous plaque burst thromboembolic ischemia formed. These plaques are thin capped vulnerable through immune responses and infl ammation and infi ltration of macrophages with intraplaque haemorrhage, neovascular expansion of the vasa vasorum, and necrotic core enlargement [64]. Evidence showed that the ROS has a double role: oxidative stress as a harmful culprit and maintaining physiological vascular homeostasis as a signalling messenger. Dysfunction occurs when the ROS level beats the maximum limit due to this disease stat formed [65].
Atherosclerosis is a lipid and dietary accession disorder, in lesion formation lipid play a role they cannot report for all atherosclerosis corporations. Atherosclerosis begins with endothelial damage, and apolipoprotein B has attractions for the basal membrane exposed in the place, where the endothelium is damaged [12].
Angiogenic expansion of the vasa vasorum in the adventitia is the key feature of the atherosclerotic process; within atherosclerotic plaques, the extensive neovascular proliferation is prominent within lesions and linked with unstable stroke, myocardial infarction, and angina. To inhibit the proliferation of the vasa vasorum and quantifying angiogenesis research used rabbit model, they combined drug delivery and molecular imaging with targeted NPs. They used the drug fumagillin with an integrin targeted paramagnetic NPS  v  3 [66].
Moreover, the cardiac magnetic resonance with α v  3 targeted paramagnetic NPs used noninvasively showed that only 1-minute dose of  v  3 with targeted fumagillin NPS lowered the angiogenesis of aortic for 3 weeks [64]. Many studies hypothesized that  v  3 with targeted NPs serve as an effi cient nanomedicine tool for drug delivery, monitoring atherosclerosis, and noninvasive characterization [66]. The application of nanozyme and their mimetic enzymes are given in Table 2.

Conclusion
Since the discovery of Fe3O4 nanoparticles as peroxidase mimics, nanozymes have attracted much attention. As natural enzyme mimics, nanozymes possess many advantages: low cost, easy preparation, excellent stability, and good durability.
They have been widely applied in sensing, environmental treatment, anti-bacterial, cancer therapy, antioxidation, etc. This review summarized the classification, catalytic mechanism, activity regulation, and recent research progress of nanozymes in these fields in detail. Although nanozymes overcome many disadvantages of natural enzymes, several exciting challenges remain. Natural enzymes are widely used in fields, including medicine, food, industry, agriculture, environment, biotechnology, etc. Compared with natural enzymes, current research on applications of nanozymes are still rather limited. These unsolved issues will be the next frontier for further applications.

Authors' contributions
Conceived and designed the experiments: Naima Nashat, and Zeshan Haider, Wrote the paper: Naima Nashat, and Zeshan Haider. Finally, fi nalized the fi nal review paper by Zeshan Haider.