In vivo General Trends, Filtration and Toxicity of Nanoparticles

The medical field is a vastly expanding one and with the discovery of nanoparticles (carbon nanotubes, diamondoids, fullerenes, gold and silver nanoparticles, quantum dots, etc.), there lies a vast field of unsolved medical diagnoses to be reassessed. Upon reassessment of the current medical problems, it is important to know what happens to a particle once it is free in the body. This review examines the different destinations of nanomaterials after they enter the body, their toxicity and their filtration. Assessing the destination of nanoparticles is done in order to find out whether they are removed by macrophages. It is concluded the strongest trends of the nanoparticles itself is of shape and surface chemistry. Toxicity of nanoparticles is found to be mostly dose-dependent. The nanoparticle filtration goal is to have the body naturally filter out the nanoparticles without a response from the immune system. *Corresponding author: G Ali Mansoori, Department of Bioengineering, University of Illinois, 851 S. Morgan St. (MC 063), Chicago, IL, 60607, USA, Tel: +1-312-996-5592; E-mail: [email protected] Received: February 27, 2013 Accepted: April 16, 2013 Published: April 29, 2013 hexagons. Fullerenes that are comprised of bonds that form 20 hexagonal ring formations and 12 pentagonal ring formations with 60 atoms of carbon are called C60 and create a ball-shaped configuration and can even be as large as C240 (240 carbon atoms) [5]. Each carbon atom is bonded to 2 other carbon atoms with single bonds. The bonds that are shared between 2 adjacent hexagonal rings observe van der Waals interactions that have stronger bond strength and are chemically written like a second bond (Figure 1 (Left)). The double bond signifies the stronger bond strength which results in the carbon atoms being physically closer together as can be seen by the bond lengths written in figure 1 (Left). It is possible to break the van der Waals “bonds” in fullerenes in order to attach other molecules or scavenge free radicals in the body [6,7]. Through this, it is possible to functionalize the fullerenes by doing things such as making them polar (and thus water-soluble) or functionalizing their size and chemistry to make them tissue-specific. Another important characteristic to the C60 fullerenes is that its diameter is 0.71 nm from nucleus-to-nucleus (entire molecule diameter, not the distance between adjacent atoms). Having it so small allows it to be highly mobile in the body and be able to pass through many different membranes in the body in order to accomplish its function (including the blood-brain barrier) [6,7]. Given that the average red blood cell is on the order of 10 μm, this allows the fullerenes to be able to even jump the membrane into individual cells, something that can be extremely useful in the new age of medicine. Carbon nanotubes (CNTs) are a sub-type of fullerene. This is a distinction based on the chemistry of the molecule. CNTs also share the hexagonal/pentagonal formation of carbon atoms with alternating double/single bonds. These molecules are of great interest in many different parts of the medical field because they have an extremely high electrical conductivity and tensile strength for their size and weight. It is due to this that CNT toxicology is reported to be of interest [8]. They are also hollow so there has been much attention paid to flow through them [9,10] and the production of them [11]. Single-walled carbon nanotubes have a wall that is only one carbon atom thick and they have, when compared to multi-walled nanotubes, higher electrical conductivity and strength per weight yet multi-walled carbon nanotubes are stronger in a one-on-one test. Multi-walled carbon nanotubes are multiple single-walled nanotubes placed concentrically around the same axis. Diamondoids generously get their name relation to diamonds due to their immensely rigid structure and density of the bonds between the carbon atoms. Diamondoid architecture is also known to be cagelike, have a self-assembly nature and be capable of functionalization [12-14]. Adamantane is a diamondoid of much interest in the medical field as it is the lowest diamondoid (least number of carbon atoms per molecule) and research of its atomic forces have revealed its uniquely high electrical conductivity [15,16]. A derivative of adamantane named memantine has been used in the pharmaceutical industry for many years (Figure 1 Right) [6,7] and is also capable of self assembly [14]. It is known to have many unique Introduction In the field of biology, there exists a series of subcategories which are independently studied and incorporated into daily life. Of these subcategories, there exists a category named nano-biotechnology. This is a category which is a cross section between fields of biology, chemistry, engineering and physics. These fields all come together to produce many different types of products that hold extremely valuable and unique characteristics [1-3] that can solve many of the most challenging biological problems of today’s world [4]. Quantum dots are a type of nanostructure comprised of a twopart core, an inner core and an outer core. These nanostructures are frequently synthesized with metal molecules for the core and are spherical in shape. They can then be polymerized with different lengths and types of polymers on their surface to both make them more biocompatible and more tissue selective. When the quantum dots become tissue selective and are targeted to cancerous tissues, they are capable of being excited via photon absorption and can elicit fluorescent resonant energy transfer (FRET) in vivo that causes lipid peroxidation and cell death. Fullerenes are a type of carbon-based molecule. Graphene, fullerenes, and carbon nanotubes (including both single and multiwalled) are comprised of carbon atoms formed in pentagons and Citation: Hartung GA, Mansoori GA (2013) In vivo General Trends, Filtration and Toxicity of Nanoparticles. J Nanomater Mol Nanotechnol 2:3. • Page 2 of 21 • doi:http://dx.doi.org/10.4172/2324-8777.1000113 Volume 2 • Issue 3 • 1000113 characteristics in response to neuroreceptors in the brain. It is also polar due to the NH2 group and extra carbon atoms. This increases the use and interest in this particle’s possibilities. Gold has much research interest in the field of cancer treatment due to its ability to be thermally excited [6,7,17-22]. Both silver and gold have been used in Alzheimer’s research previously [7]. Gold and silver nanoparticles are both considered inert and naturally hydrophobic so their passing through the body is limited. They have much potential due to their small size yet the biodistribution is still not entirely clear for either particle. This is largely attributed to attack from the immune system to such inert and non-polar particles when they have low functionalization. It is due to this that there has been much research in trying to derive a more organic version of silver nanoparticles to lower their innate toxicity [6,23,24]. Liposomes are a unique type of nanostructure that has many capabilities. It is composed of a lipid bilayer and is capable of surface functionalization and encapsulating other nanoparticles for drug delivery. It is known that they can be broken down by lysosomal enzymes within blood cells where an encapsulated drug can then take effect [25]. Due to their hydrophilic nature and surface functionalization capabilities, liposomes are highly investigated for use with tissue-specific drug delivery of toxic particles [26-38]. The first reaction of the immune system is the same no matter what type or of what nature the invader is. This is a response called the “innate” immune system response. This response is designed to kill effectively everything. It is a system of total eradication spawned by neutrophils which are designed to kill invading particles, bacteria, and even healthy cells (which could be in the process of being mutated or infected as they are being killed). Neutrophils are not the only part, but they are the main contributor to factors such as reactive oxygen species proliferation which leads to the degradation of cellular membranes through a process known as lipid peroxidation. This process leads to cell death and has been frequently consistent with in vivo findings in nanoparticle research. The second type of immune response is called “adaptive” immune response. This is the response of such items as T and B cells which are designed to kill foreign viruses outside of cells and kill cells infected and mutated by viruses/bacteria in a more specialized and unique way for each invader. Another type of adaptive immuno response, one of importance in this review, is that of the reticuloendothelial system (RES) comprised of phagocytic cells (mainly monocytes and macrophages). Phagocytes such as macrophages are cells that encapsulate foreign bodies and attempt to degrade them over time through such processes as peroxidation. The body also has liquid excretory system that filters things out of the body and digestive system to filter out what enters the blood stream if particle is ingested. These are also of high importance in this review as many particles can be filtered out naturally similar to how proteins and food are filtered. This is, of course, the easiest system which eliminates nanoparticles from the body and thus is the most favored. If a nanoparticle is to be engineered, the filtration goal is to have the body naturally filter out the nanoparticles without an inflammatory response from the immune system. The recently highlighted topics of interest by the US Environmental Protection Agency (EPA) include the toxicology of manufactured nanoparticles [39]. One of the most important factors of toxicology is biofiltration, cytotoxicity post administration. Much attention has been paid to the affects of the nanoparticles to the body but very little attention has been paid to how the body affects the nanoparticles or how the body reacts to the nanoparticles after they are in the body. The question to be asked is: do the particles get degraded by the immune system, encapsulated by the immune system, filtered out by natural filtration methods designed for waste material, or just get trapped in the body and accumulate until organ failure or death? This is a comprehensive review of the biofiltration of nanoparticles to assess the processes that the nanoparticles undergo once in the body. Advances in Nanotechnology Quantum dots It has been previously reported that fluorescent resonance energy transfer (FRET), shown in figure 2, can be an efficient and extremely useful process in quantum dot (QD) research in fighting cancerous tissues through a process called lipid peroxidation [40-42]. Regular Figure 1: (A) A single C60 fullerene created using HyperChem. The bond lengths shown are in angstroms, the shorter bonds are written as a double bond (white) to signify their increased strength and decreased proximity (1.4758 angstroms vs 1.4709 angstroms). (B) A memantine diamondoid. Blue atoms are carbon, white sticks are hydrogen atoms, and the green atom is nitrogen. A) B) Citation: Hartung GA, Mansoori GA (2013) In vivo General Trends, Filtration and Toxicity of Nanoparticles. J Nanomater Mol Nanotechnol 2:3. • Page 3 of 21 • doi:http://dx.doi.org/10.4172/2324-8777.1000113 Volume 2 • Issue 3 • 1000113 energy fluorescence can also be used to visualize tissues [43-46]. FRET is a method that can be both useful and completely toxic depending on how it is utilized. Most QDs are created via non-biocompatible organic metals and are hydrophobic [47-52]. To decrease the hydrophobicity of the QDs, in an effort to keep them from agglomerating, they are often coated. This increases their size and hinders their ability to be eliminated from the body (when over a certain size, the particles are seen as invasive as opposed to naturally occurring proteins) [53]. The effects of agglomeration, due to not coating the particles, creates entrapment into tissues like the liver [54]. The polymerization of hydrophilic ligands stops this agglomeration and maintains hydrophilicity which keeps entrapment to a minimum [54] so the increase in particle size due to polymerization still seems to be the better choice. Previous research was able to confirm the short term effects of the QD samples being different than the long-term effects (Figure 3) [47,53,54]. In the short term, the QDs accumulated in the liver, and in the long term, in the kidneys (Figure 3) [47,53]. It was also noticed that the size of the QD particles made a difference. The smaller sized particles were more readily absorbed by the kidneys and the larger ones by the liver (Figures 3-5) [47,54]. The spleen was targeted by a very select size range (5.3-5.6 nm with polyethylene glycol (PEG) coating and 2.9 nm uncoated) (Figure 5). It was also seen that there were no toxic effect after 80 days after a single intravenous (IV) injection evident in the rats, thus helping to prove that synthetic aqueous QDs can be non-toxic [47]. It was found that the largest distinction between the different metabolic processes were not based entirely on chemical interactions but rather also on size (Figure 5) [47,50,51,53,54]. The more agglomerated the particles in the body, the more likely they are to be absorbed by the liver and passed as urea [53]. This is unless they agglomerate too large in which there is no longer any metabolic function to remove them from the body and they agglomerate in tissues or get absorbed by the RES [47,54]. It was noticed that the free QDs were filtered by glomerular capillaries [55] (the capillaries at the beginning of the kidney which function to filter the blood) and excreted through urine whereas most of the QDs attached to proteins and agglomerated to larger forms were filtered through the liver [55]. Choi et al. [53] indicated that traditional, uncoated QDs are between 10 and 100 nm in diameter and have proven difficult to be cleared from the body [54]. They go further to say the QDs are frequently engulfed by the immune system [47,54]. Using transmission electron microscopy (TEM), it was verified that the core diameter (Indium-Arsenic Zinc-Sulfide core (InAs(ZnS)) was averaged at 3.2 nm [54]. PEG chains have been proven in prior research to increase solubility and delay the immune response to such particles. In this study, the lengths of PEG chains varied from 0 to 22 repeating units. An increase in the PEG chain length resulted in an increase in the blood retention time, renal excretion, and delayed extravasations from affected tissues [55]. The “shorter” chain length QDs (diameter less than or equal to 5.5 nm) were partially absorbed and cleared by the kidneys as liquid waste where they were stable however a large number of these particles were attacked by the RES system in the liver, consistent with previous research [53,54]. The “larger” chain length (diameter over 5.5 nm) QDs were absorbed and cleared by the liver into bile [54]. A noteworthy exception was the PEG8 (6.5 nm) group were found in the pancreas [54]. It was noted that if the diameter becomes too small (less than 5.5 nm), the body’s immune system attacks the molecule and attempts to degrade it using the RES as is noted in table 1. In this process, the macrophages engulf the QD and begin to eat away, or depolymerize the PEG coating, exposing the toxic core. The toxic core of the QD is then able to cause detriment to all cells it comes into contact with and no successful response by the body to facilitate its motion out of the body is present [47]. The semiconductor QDs which are made up of Cadmium-Sulfide (CdS), Cadmium-Selenium (CdSe), or Cadmium-Tellurium (CdTe) Figure 2: After the energy of a photon is absorbed by the electron and the electron is excited to a higher energy state, it becomes reactive. Upon return to ground state, the electron emits the same energy as a photon would normally have but instead of emitting it, it transfers it to an O2 molecule, creating singlet oxygen. Citation: Hartung GA, Mansoori GA (2013) In vivo General Trends, Filtration and Toxicity of Nanoparticles. J Nanomater Mol Nanotechnol 2:3. • Page 4 of 21 • doi:http://dx.doi.org/10.4172/2324-8777.1000113 Volume 2 • Issue 3 • 1000113 0 5 10 15 20 25 30 35 0.5 hours 1 hour 4 hours 15 days 80 days % ID /W (g ) heart liver spleen lung kidney intestine Figure 3: Biodistribution at different times of 2.9 nm CdTe aqueous QDs. Vertical axis is % of injected dose/weight of tissue (in grams). Data derived from previous research [47]. 0 5 10 15 20 25 30 35 40 45 0.5 hours 1 hour 4 hours 15 days 80 days % ID /W heart liver spleen lung kidney intestine Figure 4: Biodistribution at different times of 4.5 nm CdTe aqueous QDs. Vertical axis is % of injected dose/weight of tissue (in grams). Data derived from previous research [47]. 0 0.5 1 1.5 2 2.5 3 3.5 liver spleen kidney intestine bladder 4.5 nm 5.1 nm 5.3 nm 5.6 nm 6.5 nm 8.7 nm 16 nm Figure 5: Biodistribution at 4 hours post-injection of InAs core and ZnS shell QDs polymerized with different lengths of Poly-ethylene Glycol (PEG) chains. Vertical axis based on simplified data categorized by integers 0, 1, 2, and 3 corresponding to density of QDs in organ from least dense to most dense respectively [54]. have been known to emit morphological changes in cells, increase lipid peroxidation, decrease cell viability, and depress metabolic activity upon excitation or exposure of the core [47,53,54]. This is not unlike the reaction of the immune system to invasive bodies. Many of the physiological changes in the cells related to the exposure to these QDs take place in a relatively short time span (within just a few hours of the excitation, consistent with neutrophillic reactions to invasive particles) [47,53,54]. This gives credit to the belief that surface functionalization and chemistry is a very important part of the biodistribution and cytotoxicity of nanoparticles. Fullerenes A study by Baker et al. [56] that compared 3 sets of rats (control, nano-fullerenes (55 nm), and larger nano-fullerenes (930 nm referred to as the “microparticle group”)) that were exposed to an aerosol version of natural C60 fullerenes. In order to accurately create airborne Citation: Hartung GA, Mansoori GA (2013) In vivo General Trends, Filtration and Toxicity of Nanoparticles. J Nanomater Mol Nanotechnol 2:3. • Page 5 of 21 • doi:http://dx.doi.org/10.4172/2324-8777.1000113 Volume 2 • Issue 3 • 1000113 fullerenes that could be inhaled in a measurable fashion, a process was done to procure an aerosol version of the fullerenes [57] and upon x-ray spectroscopy and high-pressure liquid chromatography (HPLC), it was determined that the aerosol version of the fullerenes matched the solid state in every way. It was proven in this study that the exposure over 10 days to aerosol fullerenes did not lead to extensive toxicity in the rats [56]. The lung assessments revealed that the particles at different times in the lung decreased as a function of time implying the temporary nature of the adsorption onto the lung tissue [58]. It was also noted that for the nanoparticle group, the adsorption onto the lung tissue was 47% higher than that of the microparticle group. The deposition fraction, percent of total sample that was fixated onto the lung tissue alone, was 14.1% for nanoparticle and 9.3% for microparticle tissues (taken at 10 days) [56]. It was also noted that none of the particles in any group of rats showed traces of fullerenes in the red blood cell samples. It was also noted that the dead cell count rose in the order of control = 20nm [136-138] 4-18 nm* [136,137] Sphere Inert metal Silver All [147,150,152] 8-18 nm ***[152] Sphere Inert metal Memantine N/A**** 1 nm [133] Polar Non-Sphere Polar and unreactive PMC-16 N/A***** 1.8-2 nm [65] Polar Sphere Polar and unreactive unless specific conditions are present C3C60 N/A***** 2-8 nm [70,108] Polar Sphere Polar and unreactive unless specific conditions are present NDs N/A**** 2-8 nm [119] Polar Non-Sphere Polar and unreactive SWNT All [77,84-87] None yet Cylinder Non-Polar and unreactive unless specific conditions are present MWNT All [65,66] None yet Cylinder Non-Polar and unreactive unless specific conditions are present Liposomes <20 or >200 nm [3,175,176] 20-200 nm3 Sphere Lipid bilayer *=more or less depending on zeta potentials [123,125] **=DHLA coated ***=most useful range yet with best excretory percentages ****=natural anti-immunotoxic effects and small size disallows use to elicit toxic response *****=applications are of unique particle, size does not vary enough for immune system elicitation Table 1: The general trends of the emerging nanoparticles. The situations which cause reactions from the above listed as “unreactive unless specific conditions are met” are detailed earlier in this review. Citation: Hartung GA, Mansoori GA (2013) In vivo General Trends, Filtration and Toxicity of Nanoparticles. J Nanomater Mol Nanotechnol 2:3. • Page 6 of 21 • doi:http://dx.doi.org/10.4172/2324-8777.1000113 Volume 2 • Issue 3 • 1000113 0 1 2 3 4 5 6 7 8 Heart Liver Lungs Testes, Right control nanoparticle (55nm) microparticle (930 nm) % Dead Cell Count Figure 6: Organ weight in grams post aerosol exposure for 3 hours per day over 10 days. Vertical axis is the final percent dead cell count due to natural C60 [56]. 0 20 40 60 80 100 120 NMDA + C3 NMDA + D3 AMPA + C3 AMPA + D3 % C el l D ea th 0 μM 30 μM 100 μM 300 μM Figure 7a: Cell viability after exposure to cytotoxic media at varying concentrations. Data derived from previous research [60].