Superiorities of nanoscale materials in drug delivery

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Superiorities of nanoscale materials in drug delivery

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Iulia Ioana Lungu1,4, Alina-Maria Holban2 and Alexandru Mihai Grumezescu1,3 1

Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania 2Faculty of Biology, University of Bucharest, Bucharest, Romania 3ICUB— Research Institute of University of Bucharest, University of Bucharest, Bucharest, Romania 4 National Institute for Laser, Plasma and Radiation Physics (NILPRP), Bucharest-Magurele

1.1 INTRODUCTION Nanotechnology allows for the understanding and control of materials at the atom and molecule scales. Over recent years, several advantages have been attributed to nanotechnology, of which the following are most important: the size of the nanostructured materials have a range between 1 and 100 nm, their physical and chemical properties can be controlled through the manufacturing technique parameters at a molecular level, and the final nano-sized structures can be connected in order to attain larger constructs. Nanoscaled systems are distinguished by their particular physical, optical, and electronic properties, which have drawn attention to several areas in the field such as materials science and biomedicine. Nanotechnology has been intensely used in nanomedicine, which uses nanoscaled materials for the prevention, diagnosis, and treatment of diseases (Bamrungsap et al., 2012; Safari and Zarnegar, 2014). Essentially, nanotechnology is a field in which the manufacture of materials within the nanoscale range is possible. The most commonly used and known example is nanoparticles, which are a class of materials that, as dimensions, have less than 100 nm in at least one dimension, which, depending on the nanoparticle shape, can be 0, 1, 2, or 3D. A breakthrough in nanoparticle research was the discovery that size can critically change the physiochemical characteristics of a substance. Although considered to have a simple structure, nanoparticles actually consist of three layers: the surface, shell, and core layer. The initial layer can be functionalized with several molecules, ions, polymers, etc. Interestingly, the shell layer has a completely different chemical structure to the core layer, which is the center of the nanoparticles and is also referred to as the nanoparticle itself (Khan et al., 2017; Singh and Lillard, 2009). The idea of nanotechnology was first introduced in the early 1960s by Richard Feynman. Since then, nanotechnology has continued to grow and has been used

Materials for Biomedical Engineering: Nanomaterials-based Drug Delivery. DOI: https://doi.org/10.1016/B978-0-12-816913-1.00001-5 © 2019 Elsevier Inc. All rights reserved.

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in the research of drug delivery for several diseases. Therefore, there have been noted meaningful advancements in this field (Lobenberg, 2003). Soon after the debut of nanotechnology, controlled drug delivery systems (DDS) emerged in the late 1960s. These systems have as a purpose the precise delivery of drugs at a specific rate and period of time. Moreover, drug delivery as a field has gained increasing attention over the years due to its focus on gene and drug targeting. The aim of targeted delivery is the delivery of antibiotics to the desired location, whether it is an organ, tissue, tumor mass, etc., but at the same time without damaging the surrounding tissues (Safari and Zarnegar, 2014). Research based on nanomedicine has increased significantly over the past years and there is a current focus on commercialization. Several nanoscaled products are already on the market, with an increasing number in the pipeline. Presently, 75% of the total sales in the field on nanomedicine were accounted for by DDS (Bamrungsap et al., 2012). There is a growing demand towards “intelligent drug delivery systems” that are sensible to and can precisely counter physiopathological conditions. The use of nanoscaled constructs in these intelligent delivery systems has the capacity to react straight at the desired site, without affecting the adjacent healthy tissues and cells. The term “intelligent” refers to the specific functions of the delivery system, such as detection and repression of the diseased condition as well as targeted delivery of the drugs. Micro-sized materials have also been reported in several drug delivery applications; however, due to the similar size range of nanoconstructs with proteins and other structures found in the body, the latter are more prone to be used in these applications. Antibiotics can be encapsulated, dispersed, or absorbed by nanoparticles and this can improve the efficiency of the drug delivery system. There are several factors that can promote the outset of the therapeutic action and bioavailability, such as increased saturation solubility, enhanced dissolution, and increased adhesion to biological surfaces. Moreover, the delivery cargos can be maximized by the particle surfaces, which make nanoparticles suitable for drug delivery applications due to their high surface area. As a result, this can lead to minimization of the drug dose in order to attain the needed therapeutic activity, which furthermore can result in lowering the cost of such treatments (Bamrungsap et al., 2012; Safari and Zarnegar, 2014). As mentioned earlier, the physical and chemical features of nanoparticles can be controlled in the manufacturing stage. In addition, their size and surface properties can also be manipulated on order to attain either passive or active drug delivery. The attachments of targeting ligands to the particle surface or the use of magnetic nanoparticles are ways for achieving site-specific delivery (Bamrungsap et al., 2012; Cattaneo et al., 2010). Drug delivery with the help of nanotechnology can continue to evolve and develop new delivery systems that can extend drug markets. This can be developed for certain antibiotics that can be used for their safety and efficacy, but are not suited for clinical employment due to their poor biopharmacological features. An example is drugs that have low permeability and solubility through the intestinal epithelium, which translates into inferior bioavailability and unsatisfactory

1.2 Pharmacokinetics and Toxicity

pharmacokinetic properties. These new systems could allow drug manufacturing companies to revise the existing antibiotics in order to enhance their halflife and efficiency, therefore increasing their safety and diminishing healthcare expenses. For example, in the United States alone, there have been at least 15 new drugs approved since the early 1990s that used nanotechnology in the manufacturing of DDS (Bamrungsap et al., 2012; De Jong and Borm, 2008).

1.2 PHARMACOKINETICS AND TOXICITY There is a significant difference between the properties of a bulk material as compared to the same material at the nanometer scale. In the field of nanomedicine, the goal is to enhance the properties of nanoparticles in order to aid early diagnosis and treatment of diseases. Toxicity is a crucial aspect when discussing DDS. The toxic mechanism of bulk materials has been thoroughly investigated; however, when taking the same material and reducing it to the nanometer scale, its properties change drastically, as well as its toxicological properties (Gatoo et al., 2014; Safari and Zarnegar, 2014). In essence, there are various means through which nanoparticles can determine toxicity in the body, however the most common mechanism is the excess production of reactive oxygen species (ROS). The production of ROS is physiologically required, but in large dosages it can be potentially toxic due to induced oxidative stress and capacity to alter the physiological behavior of cells leading to cell death. There are three levels at which nanoparticle-induced oxidative stress can alter cell signaling. An initial small dosage can enhance the transcription process of defense genes, whereas a higher dosage can trigger inflammation signaling. High levels of oxidative stress have been associated with apoptosis and necrosis activation (Farokhzad and Langer, 2009; Safari and Zarnegar, 2014). Pharmacokinetics is one of the subdomains of pharmacology and is basically concerned with the absorption, distribution, metabolism, and elimination of drugs within a living system. Drugs, in regard to their pharmacokinetics, have been widely studied and their action mechanisms are relatively straightforward considering their solubility and biodistribution. However, the situation changes when discussing nano-sized materials. The pharmacokinetics of nanoparticles is challenging to understand due to their unique properties. Recent studies have suggested that the main parameters that affect the pharmacokinetics of nanoparticles are their size, shape, composition, surface charge, coating, and roughness (Barui et al., 2018; Gatoo et al., 2014).

1.2.1 ABSORPTION When nanoparticles are introduced into the body through injection, a process of formation of protein corona takes place, which is basically the coating of nanoparticles with a selected series of blood plasma proteins. However, if the

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nanoparticles are introduced through other sites before entering the blood flow, such as skin or lungs, they can accumulate supplementary molecules. As a result, the structure has a new “biological identity” that will determine a different cellular and tissue reaction. The main features that decide the interaction of the nanoparticle with the adhesive proteins are size, shape, surface charge, and solubility. The assimilation of proteins on the surface of nanoparticles greatly affects their biodistribution in the body; this can also result in alteration of the protein features. As an example, it is believed that the adsorption of fibrinogen on the nanoparticle surface stimulates the elimination of the particles through phagocytosis (Bazile, 2014; Yildirimer et al., 2011).

1.2.2 DISTRIBUTION The absorbed nanoparticles can be distributed to different organs, tissues, and cells. It can be challenging to anticipate the performance of nanoparticles in vivo. When the nanoparticles are injected directly into the bloodstream, they are rapidly eliminated by macrophages. One of the key factors in biodistribution is reaching equilibrium and organ-specific concentrations. Therefore, the permeability of the blood vessels, as well as their density, are decisive parameters; equilibrium can be reached faster in highly vascularized sites than in poorly vascularized ones. The mononuclear phagocyte system (MPS) is comprised of monocytes and macrophages. Their role is the uptake and metabolism of unfamiliar molecules. Coating nanoparticles can be a solution to avoid the MPS. As an example, PEGcoated nanoconstructs can easily evade RES uptake. Similarly, carbon nanotubes (CNTs) coated with ammonium can determine a similar outcome, but could not avoid RES uptake when coated with taurine. Surface chemistry is only one factor that can affect biodistribution, another important factor being the nanocomposite core. Even though there is no clear indicator that fullerenes or silica nanoparticles deteriorate in vivo, it has been reported that polymeric and superparamagnetic iron oxide nanoparticles (SPIONs) used as contrast agents for MRI imaging degrade in vivo (Chan et al., 2017; Khan et al., 2017).

1.2.3 METABOLISM With only a few reports on the metabolism of nano-sized materials it has been reported that enzymes could not completely metabolize inert nanoparticles; however, recent studies made on the behavior of neutrophil myeloperoxidase showed that it has the ability to degrade CNTs. Metabolization of nanostructures mostly occurs in the liver as a result of phase I and II metabolic pathways. In phase I, novel or modified functional groups are constructed through several reactions such as oxidation, reduction, or hydrolysis in order to boost the reactivity or polarity. When it comes to phase II, endogenous compounds are conjugated in order to attain increased water solubility and decreased reactivity. Generally, phase II takes place after the nanoparticles that went through phase I metabolism

1.3 Nanomaterials’ Physicochemical Properties

have attained more reactivity. Isoenzymes are the main enzymatic system of phase I metabolism; these enzymes help catalyze oxidation by electron supply transfer. Nonetheless, recent studies have shown that nanoparticles can inhibit the activity of these enzymes (Khan et al., 2017; Sahu and Hayes, 2017).

1.2.4 EXCRETION The elimination of nanoparticles from the body can occur from several routes, such as perspiration, seminal fluid, saliva, urine, etc. Studies on the elimination process undergone by single-walled CNTs with different functionalizations have shown that while those functionalized with hydroxyl were eliminated through urine in 18 days, those functionalized with ammonium did not follow liver uptake, but showed more quickly renal elimination. The stress effects of multiwalled carbon nanotubes (MWCNTs) on the liver were also examined and the results showed that even though there was agglomeration of particles in the organ, there were no negative effects registered after 28 days. By identifying the organs that get stressed by interacting with nanoparticles, a molecular basis for stress formation can be determined (Eatemadi et al., 2014; Khan et al., 2017).

1.3 NANOMATERIALS’ PHYSICOCHEMICAL PROPERTIES IN REGARD TO THEIR TOXICITY The main characteristics of nanomaterials can affect their biological behavior and therefore it is a primordial aspect to evaluate them in order to determine their toxic potential. Among these parameters, the following are worth mentioning: size, shape, composition, coating, and surface roughness (Khan et al., 2017).

1.3.1 SIZE When taking into consideration the toxic potential of nanomaterials, their size and surface area are critical because the interaction between nano-sized materials and biological systems mostly occurs at the surface of nanomaterials. There is a very firm connection between the size and surface area of nanoparticles: as the size decreases, the surface area increases. Moreover, as the size decreases, the surface becomes more reactive, increasing the potential for chemical reactions. Distribution and elimination of nanoparticles are strictly related to the size of the particles. In recent years, there has been intense research into the in vitro toxicity of various-sized nanoparticles on several cell types. It is rather difficult to determine a general idea on the toxicity mechanism of nanomaterials since each of them has unique characteristics and properties. However, the requirements for in vitro use of nanoparticles regarding their toxicity are not that strict, but those for in vivo use involve a complete understanding of the action mechanisms of the

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nano-sized materials. Currently, there is limited literature in regards to the effect of size on the in vivo behavior of nano-sized materials (Ravindran et al., 2018). Initially, the in vivo behavior of nanoparticles in the context of their toxicity was investigated in the respiratory system, since inhalation is the main path of exposure. It has been observed that there is a strict correlation between the size of the particles and pulmonary toxicity, more specifically as the nanoparticles decreased in size, pulmonary toxicity increased, regardless of the inertness of the initial bulk form of the material. Oberdoster et al. conducted a study on the effect of titanium dioxide nanoparticles of different sizes on human lungs. The results showed that when the particle size was 25 nm, as compared to 250 nm, the inflammatory response in human lungs was significantly higher (Oberdo¨rster et al., 1994). Nanomaterial biocompatibility and toxicity in vivo are highly affected by the interaction between biological systems and the mentioned nanomaterials. Studies made on mice with different kind of nanoparticles indicated that the surface area is a crucial determinant in inducing inflammatory responses in the respiratory system. Inhaled nanoparticles of different sizes, in the human respiratory system, will deposit in different sites depending on their size. For example, 100-nm or smaller nanoparticles can accumulate in all areas, 10-nm nanoparticles are usually accumulated in the tracheobronchial area, and nanoparticles that range between 10 and 20 nm agglomerate in the alveolar site. Despite the fact that size can be helpful in the evaluation of the toxic behavior of nanoparticles, there is an agreement among specialists that nanoparticle surface area or size is not the main physicochemical feature that decides their toxicity. A factor that can drastically influence the final nanoparticle size and therefore their toxicity behavior is the dispersion medium used and its afferent features (Xiaoming et al., 2015). For instance, when using a phosphate buffer, the diameter of titanium dioxide nanoparticles significantly increases as compared to the use of water. The same effect can be observed with ZnO nanoparticles. CNTs are known for their agglomeration ability due to their architecture and hydrophobic properties. There have been reports on the correlation between the size and impurities of agglomerated CNTs and their toxic behavior. It was observed that the cellular uptake procedure is dependent on the size and functionalization of CNTs. When discussing the dispersion of CNTs by intravenous injection, it was observed that they mainly agglomerate in the liver, spleen, and lungs without causing short-term severe toxicity. A study by Wick et al. showed that the toxic effect of well-dispersed CNTs is significantly lower than accumulated nanotubes. Moreover, when agglomerated, they can alter the activity and morphology of the human malignant mesothelioma cell line. Well-dispersed CNTs have both their advantages and disadvantages; one major disadvantage being that, due to their small size, when well-dispersed they promote pulmonary interstitial fibrosis. Generally, toxic behavior related to the size of nanoparticles should be investigated on each type of nanoparticle individually due to their unique properties and interactions with biological systems (Sharifi et al., 2012; Wick et al., 2007).

1.3 Nanomaterials’ Physicochemical Properties

As mentioned above, the smaller the size, the greater the surface area. The results of clinical studies showed that this aspect is related to the augmentation of ROS generation. Superoxide anions and hydrogen peroxide are formed from the interaction of nanoparticles with molecular oxygen, which furthermore results in the oxidation of molecules. The size of the nanoparticles also has a crucial impact on the rate and route of elimination from the body (Sharifi et al., 2012). Intravenously injected gold nanoparticles with sizes below 50 nm have high toxic potential because they can rapidly spread to almost all tissues, creating aggregates in the cardiovascular system, respiratory system, reproductive organs, brain, etc. However, it was reported that, when injected into mice, gold nanoparticles with sizes of 3, 5, 50, and 100 nm are not toxic, whereas those ranging from 8 to 37 nm caused an acute inflammatory response and death in a period of 3 weeks. On the other hand, when immunogenic peptides were integrated into the nanostructure, the toxic behavior was significantly reduced (Sharifi et al., 2012). The in vivo toxicity of Au and Ag nanoparticles was explored on zebra fish. Colloidal Au and Ag nanoparticles were used in different sizes: 3, 10, 50, and 100 nm. The results showed that, after 120 hours post fertilization, silver nanoparticles induced mortality that was determined by their size, whereas gold nanoparticles behaved independent of size and provoked a rate of mortality of approximately 3%. These results suggest that even though the surface area of nanoparticles has a major effect on the toxic behavior, there are additional aspects, for instance chemistry, that need to be taken into account (Sharifi et al., 2012). Amongst the first nanoparticles used as vehicles for targeted drug delivery of biological agents were polyacrylate nanoparticles, dating back to the 1970s. More recently, experiments have been carried out on polyacrylate nanoparticles synthesized from the polymerization of unsaturated monomers with sizes ranging from 40 to 250 nm. Depending on the type of monomer, the toxic behaviors were related either to the chemical properties of the nanoparticles, the size, or the molecule chain length. Commonly, it is considered that nanoparticles synthesized from biodegradable materials tend to have a lower toxic effect, as compared to nanoparticles manufactured from nonbiodegradable materials (Ren and Huang, 2010; Sharifi et al., 2012). The in vivo toxic behavior and distribution of poly(D,L-lactic-co-glycolic acid), also known as PLGA, nanoparticles were studied by Semete et al. (2010), with a size range between 200 and 300 nm. These nanoparticles were administrated to mice orally and it was observed that after 7 days almost 40% of the particles were found in the liver, and a percentage of nearly 60% of the PLGA nanoparticles were found in the brain and kidney without exhibiting any toxic behavior. It is not likely possible that these nanoparticles exhibit particle sizedependent toxic behavior due to their large size. The biological effects exhibited by biodegradable nanoparticles can be affected by the nanoparticle chemical composition and the resulted degradation byproducts. After being implanted in the body, polyesters, such as PLGA or PCL (polycaprolactone), are subjected to

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processes such as hydrolysis and enzymatic degradation that results in the formation of biologically compatible moieties: lactic, glycolic, and capronic acids (Semete et al., 2010). The nanoparticle size can affect, apart from the toxic behavior from the generation of ROS mentioned above, the degradation of the polymer matrix. The increase in surface area, determined by the decrease in particle size, allows the infiltration of physiological fluids inside the particles and enhances the degradation process (Sharifi et al., 2012).

1.3.2 SHAPE The toxic behavior of nanoparticles can also be determined by the nanoparticle shape and aspect ratio. There are several shapes that nanomaterials can possess: fibers, spheres, tubes, rings, and planes. In order to understand the shapedependent toxic behavior of nanomaterials, in vitro experiments were conducted. The shape of nanoparticles can affect the folding process of the membrane throughout endocytosis/phagocytosis. As an example, it has been indicated that spherical nanoparticles undergo the endocytosis process more rapidly than nanoparticles shaped as rods or fibers. Due to the large surface area of rod- and needle-shaped nanoparticles, the interface area with the cell membrane receptors is higher than spherical-shaped nanoparticles (Ray et al., 2009). There have been studies made on different-shaped nanoparticles on their capacity to avoid phagocytosis. The results showed that nanoparticles that have disk-like, cylindrical, or hemispherical shapes significantly surpass spherical nanoparticles in this matter; therefore the initially mentioned nanoparticles are more likely to attach to blood vessel walls, resulting in diverse biological outcomes. Studies made on mice by Radomski et at. (2005) showed that CNTs of tubular shape enhance plate accumulation and vascular thrombosis as opposed to the same nanostructures, but as fullerenes. Moreover, Park et al. (2003) observed that rod-like single-walled CNTs can obstruct potassium ion channels with efficiency three times higher than spherical fullerenes. It was reported that the length of CNTs can actually deteriorate macrophages and produce ineffective phagocytosis. As a result, the oxygen radicals and hydrolytic enzymes released from the macrophages are discharged extracellularly. Muhlfeld et al. studied the difference between long and short MWCNTs in regard to their toxic behavior. The results showed that while long MWCNTs induced an inflammatory response in the abdominal wall, short MWCNTs were efficiently phagocytized without causing inflammation. In the unfortunate event of chronic inflammation caused by bio-persistent nanoparticles, the inflammation can lead to supplementary mutagenic events that can ultimately result in the development of cancer (Muhlfeld et al., 2012). The so-called three Ds, namely, dimension, durability, and dose, have been investigated by Donaldson et al. in regard to the physicochemical properties, as well as the pathogenicity of fibers. It has been reported that long carbon fibers in the nanometric range can block stomata pores found intracranially, as well as

1.3 Nanomaterials’ Physicochemical Properties

injure endothelial and mesothelial cells. As a result of the blockage, macrophages from the pleura will agglomerate trying to phagocytize the long carbon fibers which will result in dissatisfactory phagocytosis. Moreover, the macrophages will react by discharging cytokines and oxidants that will determine an additional inflammatory response and even genotoxicity to the mesothelial cells (Donaldson et al., 2010). Allotropic materials are materials that exist in several crystal structures. These types of materials were also investigated in regard to their shape-dependent toxic behavior. An example of allotropic material would be silica: while amorphous silica has been used as a food additive and already has the approval of the FDA, crystalline silica is under continuous investigation due to the suspicion of it being a human carcinogen. Contrasting toxic behavior has also been recognized for different crystal structures of titanium dioxide nanoparticles. It has been noted that TiO2 nanoparticles in the form of rutile can cause oxidative DNA disturbance and lipid peroxidation when light is not present, whereas TiO2 nanoparticles in anatase form with the same chemical structure and size are believed to be inert. Interestingly, it was also reported that rutile TiO2 induces less ROS than anatase TiO2. Regardless of these contrary findings, all the results indicate that TiO2 in both forms, anatase and rutile, induces ROS by means of their structural architecture, which is a clear indicator of toxic behavior (Mitura and Zarzycki, 2018; Yu et al., 2017). Another example of nanoparticles that were investigated in regard to their shape-dependent toxic behavior is nickel (Ni). Different-shaped nickel nanoparticles were researched in zebra fish. With sizes of around 60 nm, arranged in aggregates, spherical-shaped Ni nanoparticles exhibited lower toxicity than dendritic clusters, results clearly indicated that the increase and decrease in toxicity can be influenced by the shape of the nanoparticles. Gold nanoparticles in spherical form, of sizes ranging between approximately 10 up to 80 nm, were observed to have a faster gold cellular uptake than gold nanorods of the same sizes. Moreover, the cellular uptake of gold nanorods attains full potential when the size of the nanorods is approximately 50 nm. TiO2 in spherical shape proved to be less cytotoxic than fibrous structures. Studies on mice of the length of TiO2 fibers have shown that 5 mm length fibers express less toxic behavior than 15 mm ones, which can also induce inflammation by alveolar macrophages (Sharifi et al., 2012).

1.3.3 COMPOSITION Even though it was implied that the size and surface area of nanoparticles have a greater efect on the overall toxic behavior, the chemical composition is more relevant when it comes to the interaction with the cell molecular chemistry and resulting oxidative stress. Using several commercially available dispersions, including TiO2, ZnO, Gd2O3, Al2O3, and other negatively and pozitively charged metal oxides, Harper et al. evaluated their toxic behavior in concern of their

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chemical composition on an embryonic zebra fish model. The oxides where injected into the embryonic zebra fish as the results were collected after 5 days of continuous waterbone exposure. The results showed that apart from yttrium oxide (Y2O3), samarium oxide (Sm2O3), and dysposium oxide (Dy2O3) none of the other metal oxides exhibited meaningful morbidity or mortality. When evaluating the toxicity of Ag2O, CuO, Al2O3, NiO, Co3O4, TiO2, and equivalent soluble salts on zebra fish, only titanium dioxide did not exhibit toxic behavior. The analyzed nanoparticles had comparable sizes, but different surface charges. However, it was conlcuded that the determinant factor in their toxic behavior was their chemical composition (Harper et al., 2008). Opposite to these findings, Chen et al. (2009) reported that mice which were injected intraperitoneal with TiO2 nanoparticles showed an acute inflammatory response. Severe injuries were caused in different organ tissues, such as lungs, liver, and spleen, where the nanoparticles accumulated (Sohaebuddin et al., 2010).

1.3.4 COATINGS Surface coatings might eliminate or mitigate the adverse effects of nanoparticles, stabilize particles, avoid agglomerations, and prevent the dissolution and release of any toxic ions. Moreover, the steric interference of surface coatings can delay the cellular uptake and accumulation of NPs, or it can facilitate NP endocytosis. Surface coating an NP could modify the surface charge or composition, leading to further toxicity due to intracellular distribution and ROS production. Many coatings are environmentally unstable or degradable and may degrade after exposing the material to biological media, therefore transforming an initially nontoxic material into a harmful one (Harper et al., 2008). Numerous studies on animals have revealed that after a large dose of ironbased NPs (2.5 mmol), after a 7-day treatment no life-threatening side effects appeared, according to histology and serological blood tests. Nevertheless, severe inflammatory and immunological responses can arise reliant on the density and type of surface coating. Generally, MNPs (magnetic nanoparticles) are coated in order to prevent the presence of free iron oxide, but the coating might be metabolized after a while. For some NPs such as quantum dots (QDs), a coating is mandatory because the shell is metallic and hydrophobic, and the core is toxic as it is composed of heavy metals, for instance, cadmium. In order to increase the core’s durability of QDs, prevent ion leaching, and increase water dispensability, a second coating is necessary. Under oxidative or photolytic conditions coatings may be unstable, therefore uncovering the metalloid core, which may lead to toxicity or lead to unexpected reactions of the QDs inside the human body. Extensively used as a coating for QDs, polyethylene glycol (PEG) is a biocompatible polymer approved by the FDA. A research group coated QDs with different molecular weight PEG (methoxy-terminated 750 Da PEG, carboxy-terminated 3400 Da PEG, and ethoxy-terminated 5000 Da PEG), and the NPs were observed for differential tissue and organ deposition in mice in a time- and size (MW)-dependent

1.3 Nanomaterials’ Physicochemical Properties

manner. The NPs coated with lower-molecular-weight PEG were eliminated from the mice’s circulations 1 hour after injection, but QDs coated with PEG 5000 remained in the blood circulation for over 3 hours. Other biocompatible polymers are extensively used as surface coating materials for SPIONs in order to stabilize colloids, deliver biologically active agents in a controlled manner, and target specific tissues by conjugation with specific ligands. Additionally, uncoated IONPs (iron oxide nanoparticles) present little solubility that can lead to precipitation during storing and also have a high rate of agglomeration under physiological conditions, obstructing blood vessels. SPION’s coatings are important, dextranmagnetite (Fe3O4) NPs cause the death of cells and reduced proliferation, which is similar to uncoated IONPs. This is attributed to the breakdown of the dextran shell exposing the cellular components to chains or aggregates of the IONPs. Xie et al. showed that coating PEG on the surface of monodisperse Fe3O4 NPs produced aggregation in vitro conditions and also reduced nonspecific uptake by macrophage cells. Although the PEGylation of Fe3O4 NPs has the power to reduce the potential of harmful biological interactions, Cho et al. found that Au NPs that have a size of 13 nm coated with PEG 5000 induce acute inflammation and eventually apoptosis in the liver of the mouse. A fairly high concentration of PEG on the NP surface does not lead to a lower NP uptake, but rather the spatial configurational freedom of PEG chains on the particle surface plays a determinant role. Coating and functionalizing CNTs may reduce their toxicity in vivo. Lacerda et al. (2006) intravenously injected MWCNTs functionalized with diethylenetriamine pentaacetic di-anhydride that resulted in stable dispersions with high excretion rates. Overall, most of the studies have indicated that coating the surface can alter the pharmacokinetics, distribution, accumulation, and toxicity of all NPs (Sharifi et al., 2012).

1.3.5 SURFACE ROUGHNESS The physical surface properties of all nanomaterials play an important role in defining the result of their interactions with cells. In contrast to specific receptor ligand interactions, for instance endocytic uptake, surface roughness along with hydrophobicity and cationic charge are the key factors that are involved in nonspecific binding forces that promote cellular uptake. Cell adhesion is promoted by minimizing electrostatic or hydrophobic hydrophilic repulsive interactions, found in NP cell interactions at nanoscale dictated by small-radii surface roughness’ strength. Particles can go through cell membranes by disrupting the phospholipid bilayer of the plasma membrane and producing momentary holes that are usually associated with cytotoxicity. Lin et al. (2010) studied the hemolytic activity of both nonporous and porous-silica NPs at different sizes. They detected that the size-dependent hemolysis effect of mesoporous silica NPs is present only when the NPs have a long-range ordered porous structure, showing that pore structure is critical in the interaction of cells and NPs. They present little cytotoxicity due to a low penetrating force of the particles through the membrane

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and fewer silanol groups on the cell-contactable surface of the mesoporous silica NPs. Angelis et al. showed that nano-porous silicon NPs with a pore size in the range of 2 nm did not present any toxicity in mouse models, as serum levels of both inflammatory cytokine IL1-b and hepatotoxicity markers LDH and GSH were standard, and there was no histological evidence of tissue pathology in the liver, kidney, spleen, lungs, and heart. Likewise, Park et al. (2009) stated that in vivo toxicity was absent, using biodegradable luminescent porous silicon NPs (Rybak-Smith, 2016).

1.4 APPLICATIONS OF NANOSCALE MATERIALS IN DRUG DELIVERY The main disadvantage of conventional drug delivery methods, such as oral administration, is the long reaction time needed for the drugs’ reactivity. Nanomaterials have the potental to be used as drug delivery carriers and in the treatment of various diseases. The main reason why nanomaterials gained so much attention for drug delivery is their physicochemical features. In order for a nanoparticle to be an effective drug delivery vehicle, it must contain three important elements: (1) a nontoxic, nonimmunogenic core material that must release the load at the specific site, (2) the therapeutic load, and (3) surface modifiers. This structure may not apply to all nanomaterials used as drug delivery carriers, but it has been generalized in order to make it easier to understand the structure of a drug delivery vehicle. Generally, the therapeutics that can be loaded onto nanomaterials are either hydrophilic or hydrophobic. Nevertheless, some nanomaterials can contain both types of therapeutics and have a potential use in multiple drug delivery (Das et al., 2013).

1.4.1 NANOSCALED MATERIALS FOR DRUG DELIVERY IN CANCER TREATMENT The main concern with classical cancer treatment approaches, such as chemotherapy, radiotherapy, and so on, is the nonspecificity of the drug action on cancer cells, affecting the surrounding healthy cells and/or tissue. Targeted drug delivery in cancer therapy can identify the dissimilarity between healthy and cancer cells, and studies have shown that the adverse effects exhibited are considerably lower than in conventional approaches. The nonspecificity of classic drug delivery translates into fast elimination of the drug, therefore the initial dose of drug must be higher, which can result in severe adverse reactions. In order to overcome these disadvantages, current research has shifted toward nanoscaled materials for targeting, diagnosis, and treatment of tumors. Due to their unique properties, nanoparticles are considered promising candidates as drug carriers. Their high surface-to-volume ratio enables the attachment of high doses of drugs which can

1.4 Applications of Nanoscale Materials in Drug Delivery

specifically target cancerous cells, without damaging the healthy ones. Moreover, the drug dose can be controlled and is significantly lower than in conventional methods, solving the major problem regarding unwanted adverse effects due to high levels of therapeutic agents (Bahrami et al., 2017). Drug delivery, generally, has two action methods: passive and active. Enhanced permeation and retention effect, also known as the EPR effect, is when the nanoparticles (approximately 400 nm in diameter) agglomerate in or in the proximity of the tumor area because of the unnatural faulty vasculature of the cancerous tissue. Passive targeting is strictly dependent on the tumor physiopathological properties as well as the physicochemical features of the nano-sized vehicles. This method also comes with disadvantages, such as ineffective drug dispersion in the required site or the fact that some tumors do not present an EPR effect. This is where active targeting is preferred—nano-sized vehicles on which tumor-specific biomarkers are attached. These carriers are internalized by the cells—a process called endocytosis—and their load is released when acidic pH is reached or by the help of enzymes. Because there is a higher chance of endocytosis taking place than the EPR effect, active targeting has been receiving more attention than passive targeting. The most common researched targeting biomarkers are folate, transferrin, epidermal growth factor receptors (EGFRs), and glycoproteins (Bahrami et al., 2017). Polymeric nanoparticles such as poly(lactic acid) (PLA) or poly(lactic co-glycolic acid) (PLGA) with sizes from 50 up to 300 nm can easily go through capillaries and be internalized by cells, therefore creating an “aggregation” of pharmaceutical agent in the desired site. PEG-PLGA nanoparticles that were loaded with paclitaxel and RGD peptides were investigated in mice that had cancer in comparison with nontargeted nanocarriers. The results showed considerable tumor growth reduction in the PEG-PLGA-based nanoparticles and prolonged life-span of these mice than the latter ones. Several other studies proposed chitosan-based nanoparticles loaded with doxorubicin for targeted cancer drug delivery (Rivera dı´az and Vivas-Mejia, 2013). Polymeric micelles have a similar action mechanism as polymeric nanoparticles. Polymeric micelles contain a hydrophobic core and a hydrophilic shell. Some therapeutic agents, such as docetaxel, are poorly water-soluble, which makes polymeric micelles the best nanocarriers for this kind of active agent. Amongst the properties of polymeric micelles the following are worth mentioning in regard to drug delivery: external factors can control the drug quantity released, small size and architecture, and increased targeting capacity achieved by functionalization. However, their main disadvantage is their low capacity for drug loading (Rivera dı´az and Vivas-Mejia, 2013). Dendrimers received attention due to their ability to solubilize hydrophobic compounds. Moreover, for cancer therapy drug delivery purposes, they can also be altered with guest molecules. Combining fluorescein and folic acid with dendrimers results in nanovehicles that can be used for imaging and healing purposes. However, clinical trials are necessary in order to determine the safety and efficacy of dendrimers (Rivera dı´az and Vivas-Mejia, 2013).

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Quatum dots (QD) are very small nano-sized crystals (from 2 to 10 nm) the size and shape of which can be accurately controlled. QDs are used as imaging agents for cancer detection due to their fluorescence spectrum. They are stable, highly sensitive, and can be conjugated with bioactive molecules. However, toxicity is a factor that needs to be carefully investigated before use (Rivera dı´az and Vivas-Mejia, 2013). Single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) have raised several concerns regarding the toxicity due to their insoluble nature in different solvents. However, this issue can be resolved by chemically modifying them and also by binding bioactive molecules. SWCNTs functionalized with PEG and paclitaxel were used on a mouse model with breast cancer and the results included tumor suppression and low adverse effects (Rivera dı´az and Vivas-Mejia, 2013). Metallic nanoparticles such as gold (Au), titanium dioxide (TiO2), and zinc oxide (ZnO) have been intensely investigated for treatment and diagnosis of cancer through drug delivery. Their main advantage is their ability to incorporate a large amount of drug due to their high surface area. The use of gold nanoparticles was studied on human cancer cells and in vivo tumor targeting. The results revealed promising outcomes together with the nontoxic and biocompatible behavior of the nanoparticles (Rivera dı´az and Vivas-Mejia, 2013).

1.4.2 NANOSCALED MATERIALS FOR DRUG DELIVERY IN CARDIOVASCULAR DISEASES Cardiovascular diseases are one of the leading causes of mortality worldwide. Therefore, there is an increasing need for the development of treatments for cardiovascular diseases. The use of biomaterials has proven to be very successful in recent years. As an example of biomaterials, coronary artery stents have been widely used to aid narrowed vessels and prevent myocardial infarction. There are several examples that can be given for biomaterials that have aided in the treatment of cardiovascular diseases; however, the future trend is the implementation of nanoscaled biomaterials that have superior properties compared to traditional biomaterials. When compared, the two have distinct properties as well as advantages and disadvantages. High surface-to-volume ratio, being carriers through blood vessels due to their size, selective cell stimulation, and functionalization in order to enter cells are all properties and actions of nanoscaled biomaterials that would be quite difficult to achieve using traditional materials (Farokhzad and Langer, 2006). Nanoscaled biomaterials have potential in several applications for the treatment of cardiovascular diseases. Surface modification of nanomaterials can be very useful in some cardiovascular practices where there is a need to promote the activity of certain cells, but at the same time suppress the activity of others. For

1.4 Applications of Nanoscale Materials in Drug Delivery

instance, one of the main disadvantages of using coronary artery stents is the appearance of neointimal hyperplasia. Enhancing the activity of endothelial cells and restraining the activity of smooth muscle cells can aid the restorative process of the endothelial layer while suppressing neointimal development (Farokhzad and Langer, 2006). Moreover, another advantage of nanoscaled materials is that they can be used as vehicles for several functional groups. These nanoscaled platforms can be used for multiple purposes all at once, for example, a single particle can exhibit features of targeting, imaging, and therapeutics. There have been recent developments in the use of nanoparticles to improve the mechanical and biological properties of currently used cardiovascular implants (Farokhzad and Langer, 2006) (Jiang et al., 2017; Table 1.1).

Table 1.1 Examples of Nanoparticles used in Cardiovascular Applications (Farokhzad and Langer, 2006) Nanoparticles

Possible Reagents

Functionalized Ligands

Potential Applications

Gold (Au)

Citrate, heparin

Peptides, genes

Silver (Ag)

Ethylene glycol/ polyvinylpyrrolidone (PVP)

Therapeutic molecules

Palladium (Pd) Platinum (Pt) Magnetite (Fe3O4) Core shell lecithin VEGF Plutonic F-127

Sodium citrate

Proteins

Enhances conductivity in cardiac patches Enhanced antimicrobial activity in heart valves Drug delivery

Polyvinyl alcohol (PVA)

Peptide

Gelation induction by core shell NPs when introduced in the functionalized ligand at physiological temp Lipidoid precipitation

Plutonic F-127

Lipidoid

Polyester carbon nanotubes (CNTs)

Nitric acid

mRNA

Used in a dispersion for the fabrication of scaffolds

Imaging contrast agent Regeneration of cardiac tissue at a specific location

Cardiovascular regeneration enhancement by gene therapy Tissue regeneration

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1.5 FUTURE PERSPECTIVES In recent decades, nanomaterials have been intensively researched for numerous biomedical applications, including targeted drug delivery, due to their unique properties such as their dimensions, biocompatibility, surface chemistry, stability in physiological conditions, and modifiable toxicity. The toxicity of nanomaterials can prove to be a challenging subject, however changing different physicochemical properties of the nanomaterials can alter their toxic behavior. Further research needs to be done on in vivo toxicity based on the physiological effect of acute and chronic adverse effects caused by nanoparticles. In order to properly and safely design future nanotechnologies, there is a need for a fundamental understanding of the interactions between nanomaterials and biological tissues.

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