An overview of nanosomes delivery mechanisms: trafficking, orders, barriers and cellular effects

Abstract

This review traces the journey of nanosomes from administration until elimination, and discusses various biological barriers. The nanosomes are imported into the body through different routes and are localized into specified organ, cell, subcellular locations or organelle compartment. The nanosomes delivery to a specific destination depends on the surface chemistry, size, shape and the presence of specific ligands. Endocytosis/exocytosis cycles are involved in the import and export of the nanosomes. The mononuclear phagocytic system and ATP-binding cassette are universal checkpoints for nanosomes trafficking. The gastrointestinal milieu is the checkpoints for orally administered nanosomes. The mucociliary escalator is a specialized obstacle for inhaled nanosomes. Dermally applied nanosomes are tackled by Langerhans cells and keratinocytes. The nanosomes intended for subcellular destinations are mainly intercepted by lysosomes. Thus, nanosomes intended for biological administration must be designed to escape various barriers. The nanosomes affect cells function by alteration of redox status, and calcium signalling, ultimately, they are exocytosed from the cells.

Keywords:

• Drug delivery
• nanosomes
• barriers
• endocytosis
• exocytosis

Introduction

Nanoscale materials have become a popular tool in the life sciences; they include natural, synthetic and hybrid materials, collectively known as nanosomes [1,2]. Although several nanosomes have already emerged in biological applications, while some of them are still under exploration [3] Nanosomes are imported into the human bloodstream through inhalation, or oral, dermal, ocular or parenteral routes. Subsequently, they are bio-distributed into specified organ, cell or subcellular locations [4]. Table 1 illustrates the common nanosomes used as drug delivery cargoes with common cargo. Drug targeting to the lymphatics, peritoneal cavity, pleural cavity, cerebral ventricles, eyes and joints represents the first order of trafficking [5]. Second-level targeting is the selective delivery of drugs to specific cell types such drug delivery to Kupffer cells in the liver or tumour cells. Third-level targeting is drug delivery specifically to an intracellular site such as organelles targeting [5]. Figure 1(A,B) shows the types and the levels of drug delivery.

Figure 1. (a) Drug delivery nanosomes can be actively targeted to specific cells by ligand labelling such as folic acid, transcobalamin, iron transport proteins, hormones and growth factors to facilitate their active cellular import. Furthermore, the cargoes can be localized in subcellular location using specific organelle localization signal. Tumour cells can be passively targeted to using effect retention and permeability effect. (b) First-level drug delivery is the targeting of nanosomes to the capillary bed of a predetermined organ or tissue (green colour). Second-level targeting is the selective delivery of drugs to specific cell types such as tumour selective drug delivery to Kupffer cells in the liver (blue colour). Third-level targeting is drug delivery specifically to an intracellular site such as organelle targeting (red colour). Fourth level is drug delivery specifically to an intra-organelles compartment (yellow colour).

Table 1. The common nanosomes (NS) used as drug delivery cargoes with common cargo.

Cargoes	Composition	Common Payload	Notes
Endosome	Endocytosis substances	Endogenous or exogenous	Endocytotic origin
Caveosome	Endocytosis substances	Endogenous or exogenous	Endocytotic origin
Exosome	Cell like structure	Proteins, and others	Cell communication, Drug cargo
Hybrid exosome	Cell like structure	Drug	Novel Drug cargo
Proteosome	Protein	Damaged proteins	Protein degradation system
Nucleosome	DNA + Protein	DNA	DNA wrapping
Genosome	DNA + liposome	DNA acting drugs	Nuclear delivery
Virosome	Viral derived	DNA acting drug	Gene therapy vector
Liposome	Phospholipids	Hydrophilic and Lipophilic drugs	Drug cargoes
Sphingosome	Sphingolipids	Hydrophilic and lipophilic drugs	Target drug cargoes
Erythrosome	RBCs like structure	Drugs	Drug cargo, biological coat
Niosome	Non-ionic surfactant, cholesterol	Hydrophilic and Lipophilic drugs	Easily prepared and stable

All of these cargoes can be targeted tumour cells or specified cell using targeting moieties such as antibodies or antibody fragments, peptides, folic acid, cobalamin, transferrin and sugars, due to the presence of receptor for such ligand in specified cell or cancer cell. Usually the ligands labelled cargoes targeted receptor mediated-endocytosis. SLNs: indicated that solid lipid nanoparticles, NLCs: nanostructured lipid carriers.

Biomembranes serve as regulatory barriers for trafficking of nanosomes between extracellular and intracellular environments [6,7]. Paracellular and transcellular transport mechanisms are involved in nanosomes trafficking between cellular compartments [8,9]. Active transport is classified into small transport and bulk active transport. In small transport, nanosomes up to 8 nm are imported into the cells via the membrane’s pores without lysosomal fusion [4]. Large trafficking of nanosomes is an energy-dependent process, and it is coordinated by internal and external signals. It is the major route for membrane trafficking of substances larger than 8 nm in diameter [10]. Endocytosis–exocytosis cycles are involved in bulk transport. The cells use such cycles to add or remove materials during feeding, division, renewal, repair, apoptosis, communication and therapy [11]. In contrast, exocytosis is implicated in the exit of material outside the cells. The substances are exocytosed outside the cells as extracellular vesicles (EVs) [9,10]. Therefore, exocytosis and endocytosis are considered crosstalk between cells.

Trafficking of nanosomes into second-level delivery (cellular delivery) and third-level delivery (subcellular delivery) depends on their surface chemistry, size and shape [4,12]. Nanosomes can induce different effects, including alteration of redox status, calcium signalling, deoxyribonucleic acid (DNA) repairing, mitochondrial dysfunction, as well as cell fragmentation [4]. In this context, nanocarriers modulate the cell cycle program, growth, differentiation, morphology and adhesion [13–17]. Finally, nanosomes are metabolized either intracellularly and used as cellular fuel or eliminated as it is, small molecules or EVs [17–20]. EVs include apoptotic bodies, microparticles and exosomes [17–20]. Trafficking of nanosomes is affected by their physico-chemical characteristics and nature of the target cell [11]. A greater understanding of nanosomes trafficking processes may lead to the development of promising multifunctional nanodevices for biomedical applications. This review article follows the journey of nanosomes from route of exposure until elimination outside the cells, in the term of trafficking, orders, checkpoints, cellular effect and obstacles of nanosomes transport.

Universal barriers of nanosomes trafficking

Universal barriers are ubiquitously distributed in different organs. They include the mononuclear phagocytic system (MPS), protein corona, ATP-binding cassette (ABC), cytochrome-P450, redox machinery and metabolizing enzymes [21]. The MPS comprises organelles and circulating macrophages whose primary function is to rid the body of foreign objects [21]. Nanosomes that enter the bloodstream are subject to rapid clearance by the MPS [19,21]. Protein coronas sequester and create multifaceted layers around nanosystems. This might result in decreasing cellular nanoparticle uptake or mitigating cell membrane damage [22,23]. The protein corona formation is influenced by the surface properties of the nanosomes [24].

ABC transporters are localized to the cell membranes, as well as organelle membranes [2]. ABC transporters couple the transport of substrates across the membrane with hydrolysis of ATP [25]. The free energy released can be utilized in the import or export of substrates from cellular compartments [25]. Therefore, ABC exporters move substrates from inside to outside of the membrane. However, ABC importers move substrates from the outside to the inside the cells [25]. ABC transporters include phosphorylated glycoprotein (PgP), breast cancer resistance proteins and members of multidrug resistance-related proteins (MRPs). Two MDR isoforms have been identified in human tissues, namely, MDR-1 and MDR-2. MDR1-encoded PgP is a major efflux transporter [2]. Thus, PgP might have a key role in the maintenance of an optimum cellular homeostatic environment [2]. ABC transporters show broad specificity for both endogenous and exogenous substrates [2].

The biotransformation barriers comprise several enzymatic reactions at specific cellular compartments to protect the cells against different materials [26]. Cytochrome P450, hydrolases, oxidases, redox machinery and others are involved in biotransformation reactions [27,28]. The products of such reactions undergo conjugation to form hydrophilic compounds that excreted in urine or bile [27,28]. Autophagy is another barrier in the regulation of the intracellular response to nanosomes. The activation of autophagic pathways prevents the accumulation of the products of oxidative stress (OS) [29].

Special barriers of nanosomes trafficking

Specific checkpoints are present in particular organs, e.g. the mucociliary escalator is present in the upper airways of respiratory system. It is contains fluids composed of phospholipids and proteins that caused nanosomes wetting and facilitate their movement towards the epithelium of bronchi [4]. The bronchial epithelial cells cilia move particles into the gastrointestinal tract (GIT) to be eliminated into the faeces. The mucociliary escalator is a predominant barrier for pulmonary trafficking of nanosomes [4]. Meanwhile, the main barriers in the GIT are the gastric and intestinal environment, mucus barrier, tight junctions, epithelial cells and subepithelial tissue [30]. Dermal barriers include keratinocytes and tight junctions that impermeable to most drug cargoes. Furthermore, melanocytes provide melanin which absorbs ultraviolet radiation and protects DNA from damage. Also, Langerhans cells, macrophages and dermal dendritic cells act as checkpoints for xenobiotics [31]. The blood–brain barrier (BBB) is surrounded by the endothelium, astrocytes and pericytes. These cells contribute to the induction tight junctions [28]. Moreover, the BBB is a transport barrier having specific transport proteins and transcytosis mechanisms that mediate the uptake and efflux of molecules [28]. Table 2 represents the checkpoints that face artificial nanosomes during their journey from the first level to the final destination.

Table 2. Checkpoints that face nanosomes (NS) during their journey from first level to final destination.

Type of barriers	Examples	Location	Escape strategies
Universal checkpoints	Protein corona, PGP, PMS, and metabolizing enzymes.	Ubiquitous distributed	NS stealth, PGP inhibition and enzymes inhibitors
Specific checkpoints	GI milieu, mucociliary escalator, BBB, stratum corneum and others.	Organs specific	Special tactics during NS fabrication
Cellular checkpoints	Tight junctions, ECM, glycocalyx and efflux transporters.	Cell membrane	CPP, ligand, very small NS and cationic NS
Intracellular checkpoints	Lysosomes, cytoplasmic crowding and binding to intracellular components	Intracellular	Endosomal escape using proton sponges, CPP, and others

First order of nanosomes trafficking

The capillary bed of predetermined target site, organ or tissue are indicated as first-level delivery such pulmonary, oral, ocular, dermal parenteral or neural trafficking.

Pulmonary trafficking of nanosomes

Inside the alveoli, few nanometer-thick barriers of epithelial and endothelial cells separate air and blood [32]. The physico-chemical characters of nanoparticles determine their trafficking in the lung. Micron-sized particles are cleared by the mucociliary escalator system, while particles less than 2.5 μm can enter the alveoli, and ultrafine particles less than 100 nm are mainly deposited in the alveolar region [33]. The mucociliary escalator is the predominant pathway for the clearance of nanoparticles that reach the lung [4]. Furthermore, lymphatic and circulatory systems aid in the clearance of inhaled nanoparticles, which are then transported to the kidneys or other organs for partial or complete elimination [4,32].

The mucociliary escalator present in the upper airways causes wetting and movement of nanoparticles towards the epithelium of bronchi. The cilia of the bronchial epithelial cells move particles from the lungs into the GIT to be eliminated into the faeces [4]. In addition, the mucus layer contains protective antioxidants, which degrade the inhaled oxidative compounds. The phagocytic removal of nanosomes occurs by the alveoli phagocytic cells, where particles are engulfed to form phagosomes that induce the chemical breakdown of the nanosomes. In the absence of alveolar phagocytosis, macrophage-loaded nanosystems are removed by the mucociliary escalator pathway [4]. Sometimes, inhaled ultrafine particles are aggregated and persist in alveoli for several days and inhibit phagocytosis, causing pulmonary toxicity [33]. Nanosomes can cross the lung epithelium and enter the blood to reach cells in the bone marrow, lymphatic system, spleen and heart [33]. Moreover, aerodynamic nanosystems can be translocated into the sensory nerve endings embedded in the airway epithelia to the central nervous system [33]. Biological coating of nanosomes with hydrophilic polymers or biomimicking decreases their macrophage clearance.

Gastrointestinal tract (GIT) trafficking of nanosomes

Nanosomes can reach the GIT directly in food, water, drug delivery nanovehicles and the mucociliary escalator pathway [33]. The epithelial cells on the surface of the intestine mediate the uptake of cargoes into the systemic circulation [4]. The intestinal epithelial cells and blood vessels are separated by several cell layers hindering the uptake of nanosomes [4,32]. The translocation of nanosystems through the intestinal barrier is a multistep process that involves diffusion through the mucus layer, contact with enterocytes, and uptake via cellular or paracellular transport [23]. Moreover, the uptake of nanoparticles occurs by the same mechanisms as those occurring in microfold cell (M cells) of Peyer’s patches [4,32]. M cells are specialized in transporting of nanosomes from the gut lumen to immune cells across the epithelial barrier [32].

The main barriers are the gastric and intestinal environment, mucus barrier, tight junctions, epithelial cells and subepithelial tissues [30]. Intestinal mucus, a complex network of highly branched biomolecules, is one checkpoint through which ingested nanosomes must pass [23]. Nanosomes with a positive surface charge are mucoadhesive, favouring penetration. However, the entry of negatively charged hydrophilic and lipophilic compounds is hindered [23]. Mucin interaction with adhesive nanosomes and larger particulates can disrupt the “bottle-brush” architecture of mucus, possibly enabling penetration upon subsequent exposures; small nanoparticles also penetrate more easily than large ones [23]. The protein networks connecting epithelial cells form three adhesive complexes: desmosomes, adherens junctions and tight junctions [34]. Such junctions act as additional intestinal barriers for nanosomes trafficking.

Nanosomes drug cargoes must overcome numerous checkpoints in the GIT. Therefore, nanosomes drug carriers should be able to shield drugs from degradation and deliver them to the intended sites within the GIT [35]. The most common mechanisms for the uptake of nanosomes into intestinal epithelial cells are endocytosis mechanisms [23]. Biodegradable nanosomes are susceptible to degradation in the GIT; therefore, the uptake of such nanomaterials is restricted after oral ingestion [4]. However, some nanosomes evade intestinal degradation and can reach the liver, kidneys and spleen. Ingested colloidal silver nanoparticles are translocated from the intestinal tract into systemic circulation [33].

Dermal trafficking of nanosomes

The skin it is directly exposed to many materials, including nanosomes. The skin comprises three layers: the epidermis, dermis and subcutaneous layer [4,32]. The epidermis is composed of a 5- to 20-μm-thick layer of keratinocytes and two layers of living cells. The underlying dermis contains hair follicles and sebaceous glands, followed by capillary vessels [4,32]. Dermal barriers, including keratinocytes and tight junctions, form a tight barrier, impermeable to most drug cargoes. Furthermore, melanocytes provide melanin, which absorbs ultraviolet radiation and protects DNA from damage [4,32]. Langerhans cells, macrophages, and dermal dendritic cells act as checkpoint for xenobiotics [31]. During the fabrication of dermal drug delivery cargoes, dermal barriers must be considered such that the nanocarriers can penetrate dermal barriers and reach the systemic circulation [31].

The stratum corneum is the rate-limiting region for nanosomes trafficking across the skin. Moreover, the physico-chemical characters of nanoparticles and flexing movement of the skin modulate nanoparticle trafficking through the skin [33]. Nanocarriers are successfully used to increase dermal penetration of therapeutics and delivery drugs to definite zones of skin [31]. Nanosomes have been found to penetrate into the human stratum corneum, epidermis and dermis, and be translocated to the systemic vasculature [33]. Nanosomes can cross-dermal barriers and reach the blood vessel system. Inside the blood, they can be transported to every part of the human body [4,32]. Topically applied TiO₂ nanoparticles have been found in the upper layers of the epidermis and in hair follicles [4,32]. Moreover, smaller (70 nm) SiO₂ nanoparticles are translocated to the lymph nodes, blood, liver, hippocampus and cerebral cortex.

Neural trafficking of nanosomes

Trafficking of drugs and nanosomes across the brain is limited by different barriers. The BBB surrounds the endothelium, astrocytes and pericytes, and these cells contribute to the induction of tight junctions [28]. Moreover, the BBB is a transport barrier that has specific transport proteins and transcytosis mechanisms mediating the uptake and efflux of molecules. Also, the brain has metabolic barriers such peptidases, cytochrome P450 and monoamine oxidases [28]. In addition, efflux transporters such as PgP, MDR-related protein, and ABC transporters limit neural trafficking [28]. The fabrication of novel drug delivery and therapeutic strategies is warranted to overcome these obstacles for the treatment of brain disease. Innovative nanoparticle design can successfully target the brain [2]. Nanosystem-functionalized drugs can act as cargoes to deliver antitumor drugs to brain tumour tissues [2].

Nanosomes reach the central nervous system through inhalation, or dermal or GIT pathways. The olfactory region of the nose is connected with the cerebrospinal fluid flow tracts around the olfactory lobe [36]. For example, inhaled silver-coated nanoparticles can reach the olfactory bulb and localize in the mitochondria of brain cells [4]. Small lipophilic cargoes introduced into the nose are distributed into the olfactory nervous system [36]. However, large molecules or water-soluble small molecules would not enter the olfactory nervous system from the nose [36]. Several studies have demonstrated that intranasally instilled ultrafine nanocargoes migrate across the olfactory nerves to the brain. Furthermore, aerodynamic nanoparticles can be translocated into the sensory nerve to the central nervous system [33].

The anionic nature of the barrier hinders the passage of most anionic nanoparticles across BBB; however, positively charged particles have greater permeability [4]. In special cases, such as hypertension and inflammation, BBB permeability is increased, allowing nanosomes access to the nervous system [4]. Various classes of nanosomes are including metallic, polymeric and lipid derived nanosomes can across the BBB and enter the brain through various endocytotic mechanisms [37].

Parenteral trafficking of nanosomes

The translocation of nanosomes following injection depends on the site of injection. Intravenously injected nanoparticles spread throughout the circulatory system, with subsequent translocation to the organs [4]. Intradermal injection leads to lymph node uptake. Intramuscular injection is followed by neuronal and lymphatic system uptake [4]. The intravenously administered functionalized nanoparticles have a longer retention time in the body compared to ingested ones. Intravenously injected nanoparticles are localized in the liver, spleen, bone marrow, lymph nodes, small intestine, brain and lungs [33]. The distribution of nanoparticles in the body is a function of their surface characteristics and size. In this regard, stealth of nanoparticles with various agents and concentrations of surfactants before injection significantly affects their distribution in the body [33]. For example, coating with hydrophilic substances prevents hepatic and splenic localization, as does the modification of nanoparticle surface with cationic compounds that facilitate arterial uptake by up to tenfold [33]. A common side effect of injecting nanosystems intravenously is hypersensitivity, a reaction that occurs in a large number of recipients and is probably due to complement activation [33]. Plasma enzymes, protein coronas and PMS are the main barriers for parenterally administered nanosomes.

Cellular nanosomes trafficking

The second level of delivery is known as cellular delivery such as liver cells or cancer cells or other cells. This level of trafficking is used in the addition or removal of materials in or out of the cells, e.g. cellular trafficking of nutrients, drugs, signals and nanosomes [7]. Different endocytotic mechanisms such as phagocytosis and nanopinocytosis are responsible for this type of nanosomes trafficking [9]. Several motor proteins, phospholipids and GTPases are involved in second-level nanosystem trafficking [38]. Small and bulk trafficking is involved in this type of trafficking. Small trafficking is a non-endocytic pathway and includes diffusion transport. This trafficking is utilized in cellular trafficking of very small nanosomes [7]. In facilitated diffusion, nanosystem transport depends on particle size and electrical charge [7]. Very small nanosomes with a diameter of less than 1 nm cross ion channels or pores in the cell membrane [4]. On the other hand, larger nanosomes are transported across the membranes by vesicles trafficking, which includes endocytosis and exocytosis processes [11]. Bulk trafficking includes clathrin-mediated endocytosis and clathrin-independent endocytosis; the main checkpoint in the cellular trafficking is the lysosomes [9]. Nanosomes delivery to cancer cells represents an excellent example of the second level of nanosomes delivery by the enhanced permeability and retention (EPR) effect [39]. By this phenomenon, the cancer cells grow quickly by production of blood vessels with wide fenestrations and defective lymphatic drainage. These factors lead to abnormal trafficking dynamics, especially for drug cargoes [39]. The EPR effect helps to transport the nanosomes and spread inside the tumour tissues by passive targeting mechanism by exploiting factors inherent to the nanosomes such as size, shape and surface charge [40]. However, active targeting depends on the presence of biorecognition molecules that attached to the surface of the nanosomes to target specific markers that are overexpressed by the cancer cells. These tactics exhibits a higher specificity and efficacy of cancer therapy [41].

Intracellular nanosomes trafficking

Intracellular trafficking represents the third level of trafficking for intracellular targeting. The Golgi complex acts as a “post office” for addressing the translated proteins [12]. Proteins are specifically delivered to cytosol, lysosomes, nucleus and mitochondria addition of specific localization signals [12]. By mimicking the function of the Golgi complex, the drug nanocarriers can be fabricated to target a particular subcellular organelle. In the intracellular environment, the endogenous cargoes are actively transported along the cytoskeletal network [42]. Therefore, assembly of nanocargoes of drugs using ligands with high affinity to motor protein enhances their transport along cytoskeletal microtubules through the cytoplasm to intended organelles [42].

Lysosomes are subcellular organelles having acidic pH and different hydrolases; they form the major checkpoint in the third level of delivery. Hereditary or acquired lysosomal dysfunctions lead to lysosomal storage diseases [43]. The lysosomal localization of drug delivery cargoes represents a hot topic in the treatment of lysosomal diseases. Lysosomal localization import has been achieved through receptors mediated (RME) using mannose-6-phosphate [44]. Cell surface receptors for folate, transferrin, vascular endothelial growth factor and low-density lipoproteins are used for lysosomal import [45]. Moreover, tyrosine-based and dileucine-based sorting signals are used for the localization of drug delivery payloads [46].

The efficient targeting of cargoes to the cytosol or subcellular organelles, the cargoes must evade lysosomal degradation [3,12]. Various strategies are ascribed to escape the cargoes from lysosomal degradation. Fusogenic peptides, pH-sensitive polymers, pH-sensitive nanoparticles and pH-sensitive liposomes are used for this purpose [3,12]. Moreover, cargoes imported by CvME are not prone to lysosomal degradation [3]. Therefore, agents facilitating lysosomal escape are viral peptides and fusogenic peptides, which undergo conformational changes in response to a change in pH to allow endosomal escape of cargoes [47,48]. Fusogenic peptides can be utilized in camouflaged nanocarriers to provoke intracellular targeting [47,48]. Bacterial peptides can induce endosomal escape and organelles targeting [42].

In addition, the proton-sponge effect is another tactic that enhances the endosomal escape of nanosomes [49]. Polyamines are cationic polymers commonly used in the construction of proton sponges with their buffering capacity [42]. An extension of the proton-sponge effect is the “umbrella effect”, this result in the protection of the nanosomes against lysosomal degradation [50].

Lipoplexes are examples of nanocargoes proposed for intracellular delivery; they are prepared from cationic lipids and are able to escape endosomes [50]. The cationic polymers can destroy biological membranes; therefore, their use has been limited [9]. Anionic carriers capable of charge reversal in the acidic endolysosome could be used instead of cationic polymers [42]. Photochemicals can also be used to disrupt the endosomal membrane upon exposure to light, either alone or incorporated into nanosomes. After light exposure, these chemicals release ROS, which destroy the lysosomal membrane. Hence, contents of cargoes are delivered intracellularly [51]. The escape of payloads from the lysosome prevents their degradation and became available in the cytosol for further delivery to subcellular compartments [51].

In such cases, nanosomes can be delivered to the mitochondria, nucleus, endoplasmic reticulum (ER), Golgi complex or other organelles. Labelling nanocargoes with a signal sequence such as nuclear localization sequences, mitochondrial localization sequences or other organelle specified signal enhances drug targeting to the organelle [13–16]. These approaches can aid the fabrication of smart nanocarriers to the targeted cytosol, mitochondria, nucleus or other organelles [12–16]. The signal peptide and mTOR can be used in the fabrication of drug nanocargoes to target the ER–Golgi network. Understanding of the ubiquitin–proteasome system (UPS) protein degradation is challenging researchers to discover promising therapeutic proteasomal targets. Bortezomib is a prototype of proteosome inhibitors. Peroxisomal drug delivery has a therapeutic value for the treatment of peroxisomal disorders [12–16]. Engineered catalase containing was fabricated and efficiently delivered into catalase-defected fibroblasts [12–16].

Mechanism of nanosomes trafficking

Vesicles and non-vesicular trafficking are involved in passages of nanosomes across biological membranes. These trafficking include active and passive diffusion. Ultrafine nanosomes are passively imported into cells without surface-specific receptors [52]. Van der Waals forces, electrostatic charges, steric interactions or interfacial tension effects facilitate cell adherence of nanosomes [7]. Charged nanomaterials cannot simply pass through the plasma membrane by diffusion. Small transport has been reported for nanosomes such as 1.47-nm diameter for organic chemicals and 4.8-nm diameter for spherical proteins [7]. Very small nanoparticles such as C60 molecules with a diameter of 0.7 nm penetrate cells via ion channels or pores in the cell membrane [4].

In case of larger nanomaterials, endocytosis is required with specific protein-coated pits, adaptor proteins and endocytic vesicles [38]. The biogenesis of endocytotic vesicles requires the action of accessory phospholipids, proteins, GTPases and dynamin and actin polymerization [38]. Endocytotic pathways mediate the transport of specific cargo to a specified location within cells. The cargoes internalized by this route are fused with early endosomes [53]. Figure 2 displays road map of nanosomes trafficking pathway during their journey.

Figure 2. Road map of nanosomes import and export across biological membranes. Abbreviations: CME: Clathrin-mediated endocytosis; CvME: Caveolae-mediated endocytosis; CCIE: clathrin- and caveolae-independent endocytosis; UPME: ubiquitin mediated endocytosis.

Machinery of nanosomes trafficking

Phagocytosis

Phagocytosis is an active and receptor-dependent import process; actin filaments play an important role in phagocytosis [54]. This path is present in epithelial cells, fibroblasts, immune cells and other phagocytic cells. Moreover, basophils, eosinophils, mast cells and natural killer cells use this mode of trafficking [54]. Physiologically, phagocytosis is an important process for cell nutrition, immune defence, tissue remodelling and autophagy [19]. Moreover, phagocytosis is specified for the degradation of foreign substances, cell debris and apoptotic bodies. Large nanosomes are phagocytosed directly, while small nanosomes are phagocytosed after opsonization [7,9]. Nanosomes less than 200 nm evade phagocytosis and have slow clearance rate. At high concentrations, they form agglomerates and are phagocytosed [4]. The macrophages then identify the opsonized nanosomes and proceed to engulf them. This internalization of nanocargo involves actin protrusions, invagination of plasma membrane and phagosome biogenesis [19]. Phagosomes can be made to contain nanosomes within an intracellular vesicle, and the phagosomes are then fused with lysosomes [3]. The endocytotic vesicles are transported from the early endosome to the late endosome and finally to the lysosome for degradation, due to the decrease in the pH and lysosomal enzymes [19]. Nanosomes must bypass the lysosomal barriers to avoid degradation [55]. Surface modification of nanosomes with hydrophilic polymers, camouflaging and mimicry are proposed as tactics to reduce nanosomes phagocytosis and lysosomal degradation [56].

Pinocytosis

Pinocytosis is the mechanism responsible for the trafficking of soluble substances [7]. Macropinocytosis occurs in specific cell types such as immature dendritic cells. This process is constitutive, non-specific and actin-dependent [6]. Normally, macropinocytosis is a typical route for the uptake of micron-size, nanosize, apoptosome, cell fragments, viruses, bacteria and nanosomes [19,54]. The internalization of particles is performed by cell membrane “ruffling”, leading to the formation of large endocytic vesicles called macropinosomes (500 nm–5 μm). Activation of tyrosine kinase receptors leads to an increase in actin polymerization and ruffling, followed by macropinosome biogenesis [54]. An example is titanium dioxide nanoparticles for the cellular import of titanium by macropinocytosis in human prostate cancer cells [19]. Macropinosomes undergo a fate similar to that of phagosome [54]. Pinocytosis is a receptor-mediated endocytosis (RME) process. It deals with the cellular entry of fluids, small molecules and nanosomes [39]. Clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME) and clathrin- and caveolae-independent endocytosis (CCIE) are the different machineries involved in pinocytosis [29]. Figure 3 displays endocytotic and exocytotic machinery of nanosystems across biological membranes.

Figure 3. Endocytotic and exocytotic machinery of nanosomes, both of CME and CvME internalize cargoes across biological membranes. In particular those labeled with folic acid, transcobalamin, transferrin, hormones and growth factors. CME is the predominant path of internalization for positively charged nanosystems. However, CvME is recommended as portal for entry for anionic nanosystems. Thus, nano-drug delivery cargoes can be actively targeted to specific cells. The CCIE pathway of nanosystem import does not depend on the presence of clathrin or caveolin in the import of nanosystems. After internalization, the vesicles containing nanosystems are recycled to the plasma membrane, or targeted to late endosomes and later to lysosomes. The cargoes labelled to organelles targeting must escape the lysosomal degradation. Finally, nanosystems are exocytosed as elements, or as natural nanocarriers in the form of exosomes, microvesicles, or apoptosome.

Clathrin-mediated endocytosis (CME)

CME is an import mechanism involved in the cellular entry of large molecules, vesicles and nanosomes. In this process, the membranes invaginate inwards with the biogenesis of clathrin-coated endocytotic vesicles ∼100 nm in size [57–59]. The vesicles are liberated from the plasma membrane with the aid of GTPases, as well as dynamin, assembled as a ring around the neck of the formed vesicles [44]. Normally, CME plays an important role in cellular signalling, membrane regeneration and entry of nutrients and pathogens [58]. Moreover, CME allows the entrance of lipoproteins, folic acid, transcobalamin, iron transport proteins, hormones and growth factors into cells [7]. Thus, nano-drug delivery cargoes can be actively targeted to specific cells. Labelling of nanoparticles with any of above-mentioned ligands facilitate their active cellular import by RME [58]. Although several studies have demonstrated that nanoparticles with sizes ranging from 50 to 300 nm can internalize cells by CME, some studies have indicated that larger particles can also be imported this mechanism [58]. The ligand-binding domain of RME has a negative charge; therefore, CME is the predominant path of internalization for positively charged cargoes. After internalization, the vesicles containing nanosomes are either guided to early endosomes, recycled to the plasma membrane or targeted to mature endosomes [57]. Later they travel to lysosomes and multivesicular bodies (MVBs) that subjected to exocytosis as natural nanocarriers in the form of exosomes, microvesicles or apoptosome.

Caveolae-dependent endocytosis

Caveolae characterized by the presence of as caveolin-1, caveolin-2 or caveolin-3. These proteins are essential in the biogenesis of caveolae [60]. Moreover, CvME contains other proteins such as cavin, which induce membrane curvature, as well as dynamin, which enables vesicle scission. Beside these proteins, vesicle-associated membrane proteins are present in caveolae [60]. Caveolin-1 promotes the binding and ordering of multiple molecules such as cholesterol, glycosphingolipids, fatty acids and membrane proteins. Thus, caveolae sequester multiple ligands responsible for cellular signalling [60]. CvME is abundant in muscles, endothelial cells, fibroblasts and adipocytes. Therefore, CvME is a promising direction for the delivery of nano-therapeutics to such cells.

CvME is a cell-specific and highly regulated cascade, signals such as nanosomes, and viruses, and other ligands initiate CvME. CvME is initiated in cholesterol-rich areas, lipid rafts or the plasma membrane [45,55]. Biogenesis of caveolar vesicles (Caveosomes) occurs by aid of dynamin and actin; the vesicles formed are in the nanoscale (60–80 nm) [60,61]. Caveosomes are flask-shaped plasma membrane invaginations (50–100 nm) with a caveolin coat [57,58]. Caveosomes are rich in sphingolipids, cholesterol and caveolin [62,63].

The caveosomes use microtubules as crossways to move in the cell, recycled back to the membrane or transported to early endosomes and fused with lysosomes. They have a neutral pH and can escape lysosomal fusion and degradation [3]. Therefore, this pathway is promising in targeting therapeutics to the intracellular organelles. CvME is recommended as a portal for entry for anionic [39]. Pathogens such as viruses and bacteria use CvME to avoid lysosomal degradation and go on to induce pathogenesis [3]. Simian virus 40 (SV40) has been reported as a prototypical ligand for CvME. Furthermore, cholera and shiga toxins are ligands for CvME. Therefore, these molecules can be used in the fabrication of nanocontainers as subcellular drug delivery cargoes [61,62]. In particular, these toxins have binding affinity towards antigens expressed on the surface of cancerous cells, making them an easy target [64,65]. Thus, the fabrication of drug nanocargoes with these toxins makes them target specific.

The CCIE pathway

The CCIE pathway of membrane trafficking does not depend on the presence of clathrin or caveolin in the biogenesis of endocytotic vesicles. Normally, the CCIE route is involved in the repair of the plasma membrane, cellular spreading and modulation of intercellular signalling [66]. CCIE includes endocytic mechanisms such as flotillin-dependent endocytosis, clathrin-independent carriers, RhoA-dependent uptake and other portals for cellular entry [66]. Actin, actin-associated proteins, GTPases and other proteins are involved in CCIE vesicle construction [66]. In addition, this pathway relies on lipid rafts and requires specific lipid compositions for vesicle generation [3]. Furthermore, dynamins play a principal part in these pathways. CCIE has been described in the trafficking of both immune cells and fibroblasts. Cargoes imported into the cell through CCIE are usually delivered to the early endosomes, followed by the transfer to late endosomes and lysosomes [54]. Moreover, CCIE-derived vesicles can be rerouted to the trans-Golgi network and recycled back to the plasma membrane or the nucleus [54,67]. Certain nanosomes have been imported by cells via CCIE and are successfully released from endocytic vesicles [44].

Cellular and subcellular effects of nanosomes

At the final destination, nanosomes can induce their cellular and organelle effects by the modulation of redox potential, calcium signalling or ion currents [4,17]. At biomembranes level, nanosomes signal transduction. Moreover, nanosomes stimulate the release of ionized calcium and free radicals from mitochondria, a high level of intracellular ionized calcium leads to the activation of protein kinase C which is involved in many intracellular signalling pathways [29]. OS can be mediated by excessive production of ROS or nitric oxide as redox messengers [29]. Such messengers induce inflammatory reactions and modify the expression of several genes in the nucleus [29]. Moreover, Cells exposed to OS induced malfunctions of the cytoskeleton and motor protein damage that affects the mechanical properties of many cell types [29]. This contributes to cell detachment, increased permeability, decrease in stability and disorganization. This induces cell retraction, rounding and deposition of massive dense filament matters adjacent to the nucleus and vacuoles in the cytoplasm [29]. The accumulation of the damaged cell debris lead to autophagy switched on. It has been reported that, incubation of cells with certain nanosomes promotes autophagy as defence mechanism [29]. Mitochondrial malfunction induces a decrease in ATP production, which is necessary for multiple cell functions, including motility and trafficking, morphological and structural integrity, division, deformation and tissue organization.

Nanosystems drug cargoes have several advantages including multiple or specific cell targeting functionalizations, and combined drug delivery [67]. Nanolipid carriers achieve brain targeting through nasal route [68]. Nanoparticles labeled monoclonal antibodies exhibit inhibitory effect on cancer cell viability through upregulation of apoptotic genes [69]. Immunohybrid nanoparticles encapsulating oxaliplatin induced time-dependent cytotoxicity, nuclear morphological changes, apoptosis and alteration of cell-cycle program [70]. Moreover, folate nanoconjugate of methotrexate showed about 40-fold increases in the concentration of methotrexate in liver after 24 h in comparison with free methotrexate [71]. Nanosomes can affecting lysosomal functions and activate autophagy, herein nanocarriers are degraded by acidic pH and lysozymes. The degradation products either are utilized in cellular activities or eliminated from the cells as small molecules, or EVs [17].

Exocytosis of nanosomes

Although the effects of nanosystems on cellular exocytotic processes have not been completely elucidated, there are a few studies suggesting nanocarriers are regulators of exocytosis. Nanosystems could be released outside the cells as EVs [11]. Furthermore, Yanes et al. [20] demonstrated that nanosystems are exocytosed outside the cells without change. Nanosystems fused with lysosomes can undergo exocytosis and release their contents outside the plasma membrane [20]. Additionally, Fröhlich et al. [9] reported that exocytosis is a mechanism for elimination of nanosystems out of cells. The physicochemical characteristics of nanosystems and nature cells influence the elimination profile of nanocarriers [20]. Although the effect of nanosystem crystallinity on exocytosis has not been completely elucidated, some studies report that amorphously formed nanosystems are rapidly eliminated from cells [20].

Nanosomes exocytosis from human colonic adenocarcinoma HT-29 cells was directly related to the extracellular calcium concentration [20]. Calcium triggers lysosomal exocytosis by changing the conformation of integral membrane proteins and enhancing the fusion of the lysosomal membrane with the plasma membrane. Conversely, cholesterol-depleting agents interfere with nanoparticle exocytosis [20]. Nanocarriers eliminated from the cells as EVs undergo endocytosis by other cells and can act as cell signalling agent [17]. Study of nanosystem exocytosis is complicated have methodology limitations, advanced techniques are needed to elucidate exocytosis. Usually nanoparticle-tracking analysis requires the combination of different techniques [11]. These techniques include elemental analysis, fluorescence microscopy and inductively coupled plasma spectrometry. Nanoparticle tracking by transmission electron microscopy can help discriminate between excretion of intact particles and ions. Single particle tracking cannot be used when particles aggregate; however, transmission electron microscopy cannot track individual particles [9]. Figure 3 displays that displays exocytotic machinery of nanosystems across biological membranes.

Conclusions

Nanosomes travel through organs, cells and organelles levels these levels are indicated as first, second and third orders of delivery. The traveling of nanosomes is limited by several barriers including MPS, PgP, protein coronas, mucin, GI milieu, mucociliary escalator and lysosomes. The fabrication strategies using hydrophilic coating, mimicry and lysosomal escape tactics aid nanosomes to evade these limitations. The localization of nanosomes at specific level depends on their physicochemical characters and presence of particular ligands. At the end of journey, nanosomes are degraded and reused or eliminated as elements or EVs. A greater understanding of nanosomes trafficking represent may aid the engineering of promising drug cargoes.

Disclosure statement

The authors declare that there are no conflicts of interest.

Additional information

Funding

The authors extend their appreciation to Kayyali Chair for Pharmaceutical Industry, Department of Pharmaceutics, College of Pharmacy, King Saud University for funding this work through the research project Number (G-2017–2).