Mitochondria, one of the vital cell organelles, is notable as being oval or bean shaped, as well as for containing a dynamic branched system that consistently divides and fuses in accordance with the regulation of fission and fusion theory 
. Mitochondria are the energy generator of eukaryotic cells, in addition to producing the adenosine triphosphate (ATP) that performs diverse functions, including the biosynthesis and degradation of protein molecules, cell division and respiration, as well as controlling membrane potential. They are adorned with countless cristae in the inner membrane of mitochondria, which are specifically involved in the generation of ATP on a massive scale. Besides the generation of ATP, mitochondria also perform numerous other tasks, such as the biogenesis of amino acids, ROS signaling, calcium ion homeostasis, apoptosis, stem cell monitoring, and the control of innate immunity 
. Further, the bidirectional drive between the nucleus and ATP generator is tightly regulated via the fission (inner membrane) and fusion (outer membrane) of mitochondria. Diverse physical platforms (including signaling pathways, protein–protein interaction, and the regulation of both calcium homeostasis and released reactive oxygen species) occur in the powerhouse of the cell. A number of metabolic processes, such as glycolysis, the Krebs cycle, oxidative phosphorylation, and acetyl CoA oxidation, are executed in the mitochondria, both for the purpose of ATP generation, as well as for dumping released electrons from the various pathways.
2. Mitochondrial Dysfunction and Diseases
Human cells comprise more than a thousand replicas of mitochondrial DNA (MtDNA). At the time of birth, these are homoplasmy that may be altered due to pathogens or mutation, and can consequently develop wild type MtDNA, or heteroplasm. The threshold of pathogenic MtDNA varies from person to person, and from organ to organ in the same individual. Abnormal mitochondria dynamics (i.e., fission and fusion), reduced oxidative phosphorylation capacity, and a transformed electron transport chain can each elicit ATP scarcity, alter ROS with poor membrane potential, and cause specific stress signaling responses. The resultant poor mitochondrial protein transportation influences enzyme assembly, which initiates several consequences, such as oxidative damage, proteotoxic stress, mutation, and the exhaustion of MtDNA. The altered morphology and functions of mitochondria can interrupt their nutrient requirements, substrate availability, and genetic signals, causing inherited MtDNA mutations which thus weaken its defense system. Figure 1 outlines the prominent features of the outer, inner, associated plasma membrane and cytosolic barriers.
Figure 1. Interconnected distinguishing features and processes occurring in the mitochondria and associated sites.
Clinical studies have revealed that progressive aging is implicated with mitochondrial impairment, and that the release and accumulation of ROS plays a crucial role in pathogenesis of age-related issues, including neurodegenerative disorders and diabetes. The imbalanced level of generation and oxidation of ROS disturbs the respiratory chain function of mitochondria, altering not only outer/inner membrane permeability, but also calcium homeostasis and high heteroplasmic MtDNA in the sufferers 
. Mitochondrial abnormality (either by MtDNA or nDNA) may occur at any age, and can affect single or multiple organs, e.g., the eye and ear. Both Leber hereditary optic neuropathy and nonsyndromic hearing loss (or deafness) have been reported as being due to abnormal mitochondrial functions. The literature reports noticeable clinical features, such as pigmentary retinopathy, cardiomyopathy, diabetes mellitus, and disorders in central nervous system (i.e., ataxia, dementia, seizures, fluctuating encephalopathy, and migraine), as being associated with the abnormal functioning of mitochondria. Figure 2
illustrates primary and secondary mitochondrial disorders and their operational issues.
Figure 2. Mitochondrial disorders (primary and secondary) and their operational issues.
Recently, Friedreich’s ataxia (a neurodegenerative defect) has been identified due to mitochondrial imbalance of ROS and reactive nitrogen species (RNS). One of the mitochondrial proteins, named frataxin, is encoded and arranged by the FXN gene, which is tremendously expressed in tissues with high metabolic rates. Frataxin is vitally involved in accumulation of the iron–sulfur clusters required for energy production. The latest evidence proposes that this protein deactivates the glutathione peroxidase enzyme, elevates thiol levels, and detoxifies the ROS associated with Friedreich’s ataxia. Hence, targeting this protein may be a possible approach for a drug delivery system 
. Genetic disorders such as Wilson’s disease, hereditary spastic paraplegia, and ethylmalonic aciduria are caused by the mutation of ATP7b, SPG7, and ETHE1 genes, respectively 
. Table 1
compiles different diseases caused by damaged mitochondria, as well as the primary methods of their management.
Table 1. Mitochondrial abnormalities and their management in different human diseases.
3. Therapeutic Approaches
Approaches such as ‘one size fits all’ and ‘precision medicine’ are often employed for the mitigation of primary mitochondrial disorders. The former manages symptomatic interventions (based on diet, exercise, and pharmacological therapeutics) that initiate mitochondrial biogenesis, provoke nitric acid synthetase, amplify ATP synthesis, control mitochondrial autophagy, and stimulate fission/fusion processes. On the other hand, the precision medicine approach focuses on specialized therapies, i.e., nucleotide supplementation, swapping of damaged MtDNA, gene replacement remedies, the elimination of noxious metabolites, and organ transplantation, if required. Table 2 compiles several therapeutic agents for the regulation of the myriad pathways involved in impaired mitochondria.
Table 2. Therapeutic agents explored for the management of impaired mitochondria.
These approaches may represent a common tactic for the management of primary mitochondrial disorders and can improve an individual’s quality of life, but they are unable to eradicate the cause of the mitochondrial disorder completely.
4. Biological Barrier and Toxicity
There are myriad barriers to designing mitochondria-targeted drug delivery, such as biological barriers and drug toxicity. After reaching to the target cell, intracellular diffusion across the outer and inner mitochondrial membrane is another challenge. Further, mitochondrial complex (I–IV) inhibition within ETC from drugs such as haloperidol and thiothixene have shown critical toxicities 
. Resultantly, neurological disorder such as Parkinson’s disease arise. Nanotechnology-based approaches are promising tools for effectively targeting mitochondria, owing to their narrow size and their efficiency in transporting bioactive agents. Both the mitochondrial membranes experience variable permeability, i.e., the outer membrane allows the permeation of a large array of small molecules, whereas the inner membrane is quite selective for transportation. The reason behind selective permeability through the inner mitochondrial membrane is the presence of specialized channel proteins there 
. Further, the transition pore of the outer mitochondrial membrane is wider than the inner mitochondrial membrane, which facilitates the faster traversal of therapeutics. The highly folded inner mitochondrial membrane is hard and possesses narrow transition slits to separate out the mitochondrial matrix within the inter membrane space. This characteristic morphology of mitochondria also poses a barrier for the passage of drug molecules across matrix. The aforesaid issues create hurdles for targeting mitochondria-based disorders. Several aspects, including high negative membrane potential (−160 to −180 mV), unambiguous protein import mechanism, variable lipophilicity, and idiosyncratic phospholipid composition of inner membrane, are a few key factors considered while designing a mitochondrion-targeted drug delivery system 
. Permeation of the outer membrane of mitochondria is concentration dependent, following passive diffusion. A negative surface charge and mitochondrial membrane potentially drive the production of ATP, in addition to exploiting for mitochondrial targeting through cationic ions and drug molecules via electrostatic interaction. Cardiolipin, another exclusive phospholipid-based component, is confined and produced in the inner mitochondria region. It plays a central role in processing several reactions and processes required for mitochondrial dynamics and respiratory chain complexes 
. Moreover, electrochemical composition and membrane potential are remarkably distinct, compared to cytoplasm. A mitochondrion comprises its own genome in form of a circular DNA containing 37 genes and 16,500 base pairs, essential for protein synthesis. The synthesized 13 proteins are basic components of the mitochondrial electron transport chain, as well as performing the process of oxidative phosphorylation 
. Mitohormesis is related to the damaged adaptive response of mitochondria, and was very first discussed by Ristow et al. in 2010. This process controls mitochondrial homoeostasis and assists organismal senescence. The literature supports that increased age favors defects in mitochondria (mitophagy), replacing new mitochondria via the process of fission and biogenesis 
. Therefore, the mitochondria’s basic functions deteriorate with aging, and can bring about several age-related disorders. In this context, drug-induced cell and mitochondrial toxicities are extensively demarcated in the geriatric population into myriad categories of pharmacological agents 
. For example, troglitazone (an antidiabetic drug) exhibited mitochondrial toxicity and was withdrawn from the market after liver dysfunction was reported. The drug potently disrupted mitochondrial-oriented respiration by inhibiting complexes II, III, IV, and V 
. Another, nefazodone (a serotonin antagonist) has been withdrawn from the market in United States in the year 2004 due to profoundly inhibiting mitochondrial respiratory chain in isolated rat liver cells 
. Biguanides (buformin, metformin, and phenformin) trigger lactic acidosis, which is directly associated with mitochondrial damage due to a surge in lactate production and respiratory inhibition 
. An over-the-counter drug, aspirin is the most prescribed medicine in elderly patients for overcoming of pain and has been reported to inhibit respiratory processes, as well as opening MPT pores and disturbing of glutathione status in mitochondria 
. Another cholesterol lowering drug, simvastatin, obstructs the ETC complexes I, IV, and V, augments calcium release, reduces mitochondrial membrane potential, and thus decreases ATP levels 
. Further, both the in vivo administration of impoverished pharmacokinetic properties and the erratic biodistribution of drugs create challenges for the design of delivery systems. In many instances, therapeutic agents lost their potency with the addition of selective coding for the targeting of mitochondria.
5. Nanoengineered Mitochondria Targeted Approaches
Nanotechnology is able to advance the pharmacokinetic and biodistribution profiles of various drug molecules without altering their pristine molecular form. The nanoformulation of drugs can modify the inherent physicochemical attributes of drugs, such as solubility, lipophilicity, half-life, and minimization of immunogenicity. Successful delivery of bioactive substances with the use of nanoparticles can be realized by customizing particle size, lipophilicity, surface charge, and the addition of specific targeting moieties. Being biocompatible and biodegradable, nanoparticles have proven potential for site-specific delivery 
, in addition to the intracellular targeting of subcellular compartments. It is evident that the architecture of mitochondria is highly distinct from other subcellular parts in eukaryotic cells. The occurrence of proton pumps in the inner mitochondrial membrane creates positive charges on the inner folded surface, whereas a negative charge is found in the mitochondrial matrix. This charge gradient develops noticeable transmembrane potential across both outer and inner mitochondrial membranes 
. Molecules such as pyruvates and lipophilic cations (i.e., triphenylphosphonium, rhodamine123, and tetrachlorotetraethylimidacarbocyanine) have been reported on for targeting mitochondria. The cationic lipoidal content of these molecules enables their permeation into the mitochondrial matrix, thus effectively targeting the region of interest. Positively charged nanoparticles induce an electrostatic interaction with the anionic phospholipids of mitochondrial membranes, and are then internalized. Inside the membrane, the nanoparticles rupture and release their drug with the mitochondrial matrix 
Recently, the cationic liposome-like vesicles known as ‘DQAsomes’ have been highly researched as mitochondria-targeted carriers, being explored to deliver cytotoxic therapeutics or DNA into the highly negative environment of cancer cells 
, as these cells experience hyperpolarized membrane potential (−220 mV) compared to the mitochondrial membrane potential of normal cells (−140 mV). Bolalipids containing DQAsomes are vesicle-like structures that are tuned for the delivery of DNA, genes, and peptides inside the mitochondrial matrix. The entrapped component is selectively transported via adorned protein pump machinery on the surface of mitochondria. DQAplexes are hybrid assemblies, comprising membrane liposomes with plasmid DNA for delivery of chemotherapeutics (specifically, into the mitochondria). Vaidya et al. conjugated folic acid on the surface of paclitaxel-entrapped DQAsomes to target overexpressive folate receptors on HeLa (tumor) cells. Outcomes obtained from confocal laser scanning microscopy revealed the enhanced antitumor activity of the designed functionalized DQAsomes, compared to bare DQAsomes 
. However, the modi operandi for restricting the precise mitochondrial targeting of this system were unclear. Other nanoengineered dendrimers with high generation number are abundantly promoted to deliver cytotoxic agents to the mitochondria. Cations (such as triphenylphosphonium, polyamidoamine, and rhodamine) containing dendrimers encompass high net-positive charges and had the ability to carry genetic materials and chemotherapeutic agents at the cell organelle 
. Table 3
summarizes the various nanodrug delivery systems utilized for the management of damaged mitochondria-based diseases.
Table 3. Nanoengineered systems for mitigation of impaired mitochondrial diseases.
Metal-based nanoparticles of sizes ranging between 5 and 260 nm were suitable for entry into the mitochondrial matrix. Wang et al. synthesized gold nanorods, in addition to discussing the comparative cellular internalization and intracellular trafficking through the mitochondria, lysosomes, cytoplasm, and endosomes of both normal and cancerous cells. A seed-mediated growth method was utilized to design cetyltrimethylammonium–ammonium bromide wrapped gold nanorods. The average particle size and zeta potential of developed nanorods were 18 ± 0.5 nm and +29.3 ± 0.7 mV, respectively. The prolonged retention and cationic surface charge of the nanorods reduced the mitochondrial membrane potential and generated reactive oxygen species in the mitochondria of lung cancerous cells (A549 cells), which caused apoptosis or cell death 
. Figure 3
outlines the principal mechanism of metallic nanoparticles for targeting mitochondria.
Figure 3. Schematic illustration on the mechanisms of metallic nanoparticles for targeting different pathway signals of mitochondria. PI3K: Phosphatidylinositol 3 Kinase; mTOR: Mammalian Target of Rapamycin; AMPK: Adenosine Monophosphate Activated Protein Kinase; Erk: Extracellular Signal-Regulated Kinase; Akt: Protein Kinase B; MAPK: Mitogen Activated Protein Kinase; ULK1: 112-kDa protein; BNIP3/NIX: Bcl-2 interacting protein 3; PINK1/Parkin-PTEN-induced kinase 1/autoinhibited E3-Ub ligase; SQSTM/P62: Sequestosome 1/p62 scaffold protein; NBR1: Ubiquitous Scaffold Protein; LC3-II: Ubiquitin-Binding Domain (Phosphatidylethanolamine conjugate).
However, the toxicity of metallic nanoparticles is reliant on biophysical characteristics, such as size, surface area, charge, and aggregation. These features affect biodistribution and internalization within organ systems, in addition to changing molecular interactions with receptors or macromolecules 
. Published reports have suggested that there is a direct correlation between the particle size of nanoparticles and their efficiency to generate ROS in vital organs. For instance, silver nanoparticles with 10 nm size displayed superior biodistribution and caused more fatal effects in the liver and spleen cells, compared to the size range 40–100 nm 
. As with the nanoparticle size, shape also affect biodistribution and clearance from the body. Long fibrous metallic nanoparticles exhibited more serious effects and were difficult to remove out from organ systems 
. Similarly charged nanoparticles show greater accumulation in the target site compared to the noncharged ones. Peak et al. investigated the effects of particle size and surface charge of zinc oxide nanoparticles on pharmacokinetic parameters, including biodistribution and clearance upon administration of a single oral dose to rats. It was evident that negatively charged zinc oxide nanoparticles were extensively absorbed by the systemic circulation, compared to those that were positively charged. Zinc oxide particles of 20 nm were swiftly eliminated through the biliary and fecal routes