Effects of Nanoparticles on the Immune System

The effects of nanoparticles on the immune system and potential mechanisms underlying these effects.

Introduction:

Nanoparticles are increasingly gaining popularity in consumer goods, medical products, electronics, and optics within the last 20 years. Nanoparticles range in size from 1 to 100 nanometers and are categorized by surface area, agglomeration ability, production of contaminants, surface charge, and crystal structure (Frenkel, Yevick, Cooper, & Vasic, 2011). They can be found naturally in the environment as well as engineered. Common engineered nanomaterials such as nanosilver, carbon nanotubes, nanogold, and titanium oxide nanotubes are in over thousands of consumer goods and used in many occupational settings (Nel et al., 2009). For example, nanosilver is widely used for its microbial properties while nanogold is used in optics and drug delivery (Vance et al., 2015). Those mentioned were only a select few, but many types of engineered nanoparticles are composed of different types of metals, polymers, lipid, protein based, and carbon based (Frenkel et al., 2011). Major advancements in the fields of physics and chemistry has given rise to many different engineered nanoparticle structures where they can be used in drug delivery and therapeutics, biological sensors, and targeted molecular imaging (Buzea, Pacheco, & Robbie, 2007). While the amount of consumer products that are utilize nanoparticles is exponentially increasing, this raises the question of adverse health effects on humans as well as persisting effects on the environment(Vance et al., 2015). However, while there are many benefits that arise from the usage of nanoparticles, many studies have found a common theme between nanoparticle exposure and adverse immunological responses such as but not limited to inflammation.

The immune system is our body’s line of defense as it protects us from foreign particles and pathogens, externally and internally. In toxicology, the immune system is one of the most important systems when determining toxicity. Exposure to nanoparticles has the ability to activate the immune system, being a foreign entity, the nanoparticle will either be cleared or accumulate in a specific location of the body(Fifis et al., 2004). What determines the degree of immune response solicited by a nanoparticle is its physicochemical properties (Fifis et al., 2004). Coating and charge of a nanoparticle can play a major role in activation as well as size and surface area (Aggarwal, Hall, McLeland, Dobrovolskaia, & McNeil, 2009). It has been demonstrated that size of the nanoparticle plays an active role in immunological response and toxicity upon cellular uptake by macrophages and dendritic cells (Manolova et al., 2008). Size of the nanoparticle can play a complicated role when macrophages will attempt to phagocytize a particle with a high aspect ratio (Manolova et al., 2008). The high aspect ratio will lead to frustrated phagocytosis by the macrophage which has the potential to result in inflammation (Manolova et al., 2008). While size and surface composition influences uptake, surface charge plays a major role in immune activation as well as membrane and uptake activities (Frohlich, 2012). Comprehending how nanoparticle’s physicochemical properties influence cellular interactions leading to an immune response is still an obstacle but critical in the future of nanomedicine and drug delivery.

Many previous studies have elucidated the effects of nanoparticles on activating the immune system. None the less more ongoing studies are uncovering that immunosuppression is a result of nanoparticle exposure. With the growing uses of nanoparticles, immunoactivation and immunosuppression can either be beneficial or detrimental. These effects have been heavily studied and have highlighted major key findings in the benefits in cancer treatments as well as the disadvantages in autoimmune disorders.

Nanoparticles Activating the Immune System:

Nanoparticles may be recognized by the immune system as a possible foreign invader activating it, creating a cascade of undesirable events. In this next section, we will review how nanoparticles activate the complement system as well as triggering hypersensitivity immune reactions causing inflammatory responses.

Complement activation by nanoparticles

The complement system is part of the innate immune system. This system heightens the responses of phagocytic cells such as macrophages and increases antibody production in response to foreign entities such as pathogens and damaged cells (Moghimi et al., 2011). The compliment system is made up of small pro proteins that when stimulated will activate proteases to cleave specific proteins beginning the complement cascade (Szebeni et al., 2007). The cascade once activated can cause opsonization, followed by cleavage of proteins, stimulating cytokine release, and ultimately resulting in release of chemotactic molecules and anaphylatoxins which can result in fatality (Szebeni et al., 2007). When nanoparticles are introduced into the body they can induce complement activation. Studies have shown when nanoparticles induce complement, this can result in rapid clearance of the nanoparticle by compliment receptor mediated phagocytosis (Markiewski & Lambris, 2009).

Complement activation by nanoparticles can also cause hypersensitivity and result in anaphylaxis, as previously mentioned. This hypersensitivity can be avoided if not a desired effect because one of the many benefits that nanoparticles possess is the ability to engineer their outer surface to have the desired benefits needed (Frohlich, 2012). Surface properties need to be tested prior to administration of the nanoparticles to determine what level of activation is solicited. In addition to determine activation, decreasing possible interactions to lessen hypersensitivity (Frohlich, 2012). Activation of the complement system by nanoparticles is not always disadvantageous. A benefit to activating the complement system is that it has the ability to increase antigen presentation which would increase the efficacy of nanoparticles being administered via vaccination subcutaneously or intradermally (Balenga et al., 2006).

Another way that nanoparticles can activate the complement cascade is by the formation of a biomolecular corona (Monopoli, Aberg, Salvati, & Dawson, 2012). A biomolecular corona alters the physicochemical properties of the nanoparticle, such as size and interfacial composition (Monopoli et al., 2012). The biomolecular corona is a coat on the nanoparticle enabling the cell to see and recognize different macromolecules on the nanoparticle’s surface (Monopoli et al., 2012). The formation of a biomolecular corona is the adsorption of proteins, lipids, carbohydrates, and nucleic acid attached to the surface of a nanoparticle. The formation of a biomolecular corona can occur naturally in the blood or can be engineered. A few mechanisms have been proposed elucidating the ways complement activation occurs upon biomolecular corona formation in the body (Chen et al., 2017). The biomolecular corona formation can make the nanoparticle vulnerable to a C3b attack which is a complement 5 convertase in the complement cascade that is able to initiate the assembly of the membrane complex attack (Chen et al., 2017). A study done by Chen et al., was able to show after serum and plasma proteins were able to bind the nanoworm (a type of nanoparticle where shape resembles a worm) forming a biomolecular corona, C3 will covalently bind to these proteins in vitro (Chen et al., 2017). These surface bound proteins would cause a rapid assembly of complement proteins. When done in vivo the nanoworms were able to activate an alternative complement pathway in animal blood indicative of a longer circulating half-life in the body resulting in a continuous inflammatory response if not cleared (Chen et al., 2017). Although not extensively studied, the compliment cascade is a major molecular pathway that the nanoparticle activates creating an immune response.

Nanoparticles stimulating an inflammatory response

Innate immunity is comprised of cells that express receptors that either recognize pathogen-associated molecular patterns (PAMPs) or danger-damage associated molecular patterns (DAMPs) (Chen et al., 2017; Dobrovolskaia, Aggarwal, Hall, & McNeil, 2008). PAMPs work through a specific kind of pattern recognition receptor such as toll-like receptors (TLR) which has the potential to distinguish microbial structures (Dobrovolskaia et al., 2008). Nanoparticles may be linked to activation of DAMPs or PAMPs, which has the ability to cause cellular response resulting in reactive oxygen species (ROS), cell injury, and accelerated inflammation (Fadeel, 2012). These responses are not always unwanted. Pattern recognition receptors (PPRs) are located on macrophages and dendritic cells in turn will generate the production of type I IFNs (Luo et al., 2017). There are a variety of PPRs that activate IFNβ such as TLR4 receptor, cytoplasmic dsRNA responsive retinoic acid- inducible gene- 1 (RIG-I), and the latest identified Stimulator of Interferon Gene (STING) (Luo et al., 2017). Activation of the STING pathway with nanoparticles is being used as a novel approach in potential therapeutics for tumor treatments (Sokolowska & Nowis, 2017).

Activation of the inflammasome has been extensively studied in regard to nanoparticle exposure. Silver nanoparticles can cause inflammasome activation via the breakdown of transcription factor 6 (ATF-6) while titanium oxide can serve as a potassium channel agonist activating the inflammasome causing an abundance of ROS (Demento et al., 2009; Simard, Vallieres, de Liz, Lavastre, & Girard, 2015). Although much research has established that nanoparticles can cause inflammation through PPRs then causing activation of the inflammasome, this research is still at its beginnings and more about these mechanisms need to be further explored.

Plasma protein interactions and macrophage phagocytosis

The diverse physicochemical properties of the nanoparticles as spoken previously has a major influence on whether the macrophage will uptake and clear the particles. Macrophages will phagocytize cationic or anionic particles and larger than smaller particles more efficiently (Zahr, Davis, & Pishko, 2006). Polyethylene glycol (PEG) is a polymer coating commonly used in nanoparticle drug delivery to help avoid immune recognition (Ishida, Wang, Shimizu, Nawata, & Kiwada, 2007). Avoiding the immune system will allow the nanoparticle to circulate the blood stream longer. PEG has shown a significant decrease in macrophage uptake as well as liver phagocytes (Ishida & Kiwada, 2008). Utilization of PEG will increase efficacy of nanoparticles to make it to their therapeutic destination without the majority of them being eliminated. The reason why PEG nanoparticles are efficient is because it will create a steric shield preventing plasma proteins from binding to the surface of the particle causing opsonization which can trigger the complement cascade (Ishida & Kiwada, 2008; Kim, El-Shall, Dennis, & Morey, 2005).

When nanoparticles do not contain PEG, it will undergo opsonization. When plasma proteins bind onto the surface of nanoparticles it changes the initial intended properties of the nanoparticle (Owens & Peppas, 2006). Opsonization can affect absorption, distribution, metabolism, and clearance. In addition of plasma proteins to the surface of the nanoparticles, nanoparticles will affect macrophage uptake (Owens & Peppas, 2006). The reason for affecting macrophage uptake is because the surface of the nanoparticles when it undergoes opsonization in the blood will have a different surface charge and different size. The mechanism of protein adsorption remains unclear but studies have indicated that the most common blood proteins to adhere to a nanoparticle are immunoglobulins, proteins that make up the complement system, fibrinogen, and albumin (Frank & Fries, 1991). It can be concluded that one of the main reason for the adherence for these surface proteins is to mark the nanoparticle for rapid clearance from the body either through macrophages, complement cascade, or alternative pathways.

Antigen presenting cells (APCs) are the main initiators of the adaptive immune response (Mottram et al., 2007). Nanoparticle exposure can alter this mechanism drastically causing increased immunomodulation. Macrophages and dendritic cells are categorized as APC’s.  In a study where dendritic cells were exposed to titanium oxide and silicon oxide nanoparticles caused an immediate activation of the inflammasome (Winter et al., 2011). In addition to the inflammasome activation there was increased expression in CD80, a protein found on dendritic cells and activated B and T cells, as well as an increase in the major histocompatibility complex MHC-II found when dendritic cells are activated (Winter et al., 2011). Comparably, zinc oxide nanoparticles have also shown to play a role in adaptive immunity. Zinc oxide will heighten the production of T helper cell type 2 (Roy et al., 2014). This intensified reaction was shown in an ovalbumin model of murine allergic asthma where there were major increases in antibodies IgE and IgG and increased T-cell proliferation (Roy et al., 2014). Thus far we have seen nanoparticle’s play a role in the activation of both innate and adaptive immunity through various mechanisms.

Nanoparticles stimulating allergic reactions

Recent studies have been able to link nanoparticle exposure, such as single and multi-walled carbon nanotubes and allergic reactions. The acute inflammatory response mediated by these nanoparticles are commonly seen in occupational settings more specifically in the manufacturing of materials containing nanoparticles and even the manufacturing of the nanoparticles themselves (Mitchell, Lauer, Burchiel, & McDonald, 2009). From toxic epidermal necrolysis to simple dermatitis these immune responses have been seen with nanoparticles not intended for medicinal use (Toyama et al., 2008). This brings awareness to the fact that in an occupational setting these particles can have damaging effects. With the growth of new particles exponentially increasing and not being regulated, this is a warning that more precaution that needs to be taken in the making and handling of these nanoparticles in the occupational setting.

Mechanisms of these immunostimulatory reactions caused by nanoparticles occur through the release of different inflammatory cytokines. Many studies have implicated certain nanoparticles such as gold colloids and lipid nanoparticles in the release and activation of cytokines (Niidome et al., 2006). Emerging studies have also suggested that in production of certain nanoparticles the surfactants used or bacterial endotoxins present can mediate allergic reactions upon exposure to the immune system (Scholer et al., 2001).

Nanoparticle’s role in Immune Suppression

In toxicology when confronted with exposures what is typically researched are what pathways are activated and causing a desired or undesired effect. In the field of nanotoxicology there is a surplus of information on immunoactivation but the amount of information on immune suppression is few and far in between. Other than nanoparticles used for the delivery of immunosuppressive drugs very few studies have really focused on parts of the immune system that just simply isn’t active upon exposure (Kalkanidis et al., 2006). An inhalation study focused on multi-walled carbon nanotube exposure and was able to demonstrate B-cell function was suppressed through TGF-b produced by alveolar macrophages (Mitchell et al., 2009). Other desired effects that stem from immunosuppression is sometimes hypersensitive is inhibited and this could aid in creation of new therapeutics. More research is needed in the field of nanoparticles and its role and mechanistic pathway in immune suppression.

Bridging the gaps in research & Conclusion

Growing in applications, nanoparticles are in consumer goods, health and medical products, as well as in occupational settings. Although there are many uses, research is behind in elucidating the health concerns and adverse effects of nanoparticles upon exposure. Many of the studies mentioned in this review are in its infancy due to the number of new nanoparticles engineered annually. There is a major disconnect with nanoparticles being manufactured and used in an occupational setting. More studies need to be done in occupational settings. It needs to be further investigated how immune activation is playing a role in people that are coming in contact with raw nanoparticles daily. Recent work has demonstrated that nanoparticles do effect the immune system but the main molecular mechanism still remains unknown. With the number of new nanoparticles being produced annually, it is crucial that characterization be done on them. Without physicochemical properties being known and lack of characterization, it is nearly impossible to foresee the biological effects that they have on the body. In order to increase our knowledge and bridge the gaps, other immunological pathways need to be tested. The STING pathway is one of the few recently discovered. Designing experiments that look at immune suppression not just as a vehicle for drug delivery but as a reaction to the nanoparticles themselves is detrimental. When nanoparticles are created, they should be immediately characterized and there interactions in vitro should be tested prior to use. Ongoing studies should follow in vivo and in humans to determine if the effects seen in vitro will translate to animal and human studies. Complement activation should still be looked at, targeted delivery systems, RBC hemolysis, and granulocyte activation are all factors that play a major role in the immune system that need to be better understood in regard to nanoparticle exposure. All in all, our understanding of nanoparticles in the immune system has increased throughout the years but there is a vast amount of room for growth and research. We have just begun to touch the surface of some of these topics, more future mechanistic studies and collaborations with chemist, physicist, biomedical sciences are required to get a full understanding on nanoparticles and its impact on the immune system.

Bibliography

Aggarwal, P., Hall, J. B., McLeland, C. B., Dobrovolskaia, M. A., & McNeil, S. E. (2009). Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv Rev, 61(6), 428-437. doi:10.1016/j.addr.2009.03.009

Balenga, N. A., Zahedifard, F., Weiss, R., Sarbolouki, M. N., Thalhamer, J., & Rafati, S. (2006). Protective efficiency of dendrosomes as novel nano-sized adjuvants for DNA vaccination against birch pollen allergy. J Biotechnol, 124(3), 602-614. doi:10.1016/j.jbiotec.2006.01.014

Buzea, C., Pacheco, II, & Robbie, K. (2007). Nanomaterials and nanoparticles: sources and toxicity. Biointerphases, 2(4), MR17-71.

Chen, F., Wang, G., Griffin, J. I., Brenneman, B., Banda, N. K., Holers, V. M., . . . Simberg, D. (2017). Complement proteins bind to nanoparticle protein corona and undergo dynamic exchange in vivo. Nat Nanotechnol, 12(4), 387-393. doi:10.1038/nnano.2016.269

Demento, S. L., Eisenbarth, S. C., Foellmer, H. G., Platt, C., Caplan, M. J., Mark Saltzman, W., . . . Fahmy, T. M. (2009). Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy. Vaccine, 27(23), 3013-3021. doi:10.1016/j.vaccine.2009.03.034

Dobrovolskaia, M. A., Aggarwal, P., Hall, J. B., & McNeil, S. E. (2008). Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Mol Pharm, 5(4), 487-495. doi:10.1021/mp800032f

Fadeel, B. (2012). Clear and present danger? Engineered nanoparticles and the immune system. Swiss Med Wkly, 142, w13609. doi:10.4414/smw.2012.13609

Fifis, T., Gamvrellis, A., Crimeen-Irwin, B., Pietersz, G. A., Li, J., Mottram, P. L., . . . Plebanski, M. (2004). Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol, 173(5), 3148-3154.

Frank, M. M., & Fries, L. F. (1991). The role of complement in inflammation and phagocytosis. Immunol Today, 12(9), 322-326. doi:10.1016/0167-5699(91)90009-I

Frenkel, A. I., Yevick, A., Cooper, C., & Vasic, R. (2011). Modeling the structure and composition of nanoparticles by extended X-ray absorption fine-structure spectroscopy. Annu Rev Anal Chem (Palo Alto Calif), 4, 23-39. doi:10.1146/annurev-anchem-061010-113906

Frohlich, E. (2012). The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomedicine, 7, 5577-5591. doi:10.2147/IJN.S36111

Ishida, T., & Kiwada, H. (2008). Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes. Int J Pharm, 354(1-2), 56-62. doi:10.1016/j.ijpharm.2007.11.005

Ishida, T., Wang, X., Shimizu, T., Nawata, K., & Kiwada, H. (2007). PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner. J Control Release, 122(3), 349-355. doi:10.1016/j.jconrel.2007.05.015

Kalkanidis, M., Pietersz, G. A., Xiang, S. D., Mottram, P. L., Crimeen-Irwin, B., Ardipradja, K., & Plebanski, M. (2006). Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity. Methods, 40(1), 20-29. doi:10.1016/j.ymeth.2006.05.018

Kim, D., El-Shall, H., Dennis, D., & Morey, T. (2005). Interaction of PLGA nanoparticles with human blood constituents. Colloids Surf B Biointerfaces, 40(2), 83-91. doi:10.1016/j.colsurfb.2004.05.007

Luo, M., Wang, H., Wang, Z., Cai, H., Lu, Z., Li, Y., . . . Gao, J. (2017). A STING-activating nanovaccine for cancer immunotherapy. Nat Nanotechnol, 12(7), 648-654. doi:10.1038/nnano.2017.52

Manolova, V., Flace, A., Bauer, M., Schwarz, K., Saudan, P., & Bachmann, M. F. (2008). Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol, 38(5), 1404-1413. doi:10.1002/eji.200737984

Markiewski, M. M., & Lambris, J. D. (2009). Is complement good or bad for cancer patients? A new perspective on an old dilemma. Trends Immunol, 30(6), 286-292. doi:10.1016/j.it.2009.04.002

Mitchell, L. A., Lauer, F. T., Burchiel, S. W., & McDonald, J. D. (2009). Mechanisms for how inhaled multiwalled carbon nanotubes suppress systemic immune function in mice. Nat Nanotechnol, 4(7), 451-456. doi:10.1038/nnano.2009.151

Moghimi, S. M., Andersen, A. J., Ahmadvand, D., Wibroe, P. P., Andresen, T. L., & Hunter, A. C. (2011). Material properties in complement activation. Adv Drug Deliv Rev, 63(12), 1000-1007. doi:10.1016/j.addr.2011.06.002

Monopoli, M. P., Aberg, C., Salvati, A., & Dawson, K. A. (2012). Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol, 7(12), 779-786. doi:10.1038/nnano.2012.207

Mottram, P. L., Leong, D., Crimeen-Irwin, B., Gloster, S., Xiang, S. D., Meanger, J., . . . Plebanski, M. (2007). Type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus. Mol Pharm, 4(1), 73-84. doi:10.1021/mp060096p

Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P., . . . Thompson, M. (2009). Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater, 8(7), 543-557. doi:10.1038/nmat2442

Niidome, T., Yamagata, M., Okamoto, Y., Akiyama, Y., Takahashi, H., Kawano, T., . . . Niidome, Y. (2006). PEG-modified gold nanorods with a stealth character for in vivo applications. J Control Release, 114(3), 343-347. doi:10.1016/j.jconrel.2006.06.017

Owens, D. E., 3rd, & Peppas, N. A. (2006). Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm, 307(1), 93-102. doi:10.1016/j.ijpharm.2005.10.010

Roy, R., Kumar, S., Verma, A. K., Sharma, A., Chaudhari, B. P., Tripathi, A., . . . Dwivedi, P. D. (2014). Zinc oxide nanoparticles provide an adjuvant effect to ovalbumin via a Th2 response in Balb/c mice. Int Immunol, 26(3), 159-172. doi:10.1093/intimm/dxt053

Scholer, N., Olbrich, C., Tabatt, K., Muller, R. H., Hahn, H., & Liesenfeld, O. (2001). Surfactant, but not the size of solid lipid nanoparticles (SLN) influences viability and cytokine production of macrophages. Int J Pharm, 221(1-2), 57-67.

Simard, J. C., Vallieres, F., de Liz, R., Lavastre, V., & Girard, D. (2015). Silver nanoparticles induce degradation of the endoplasmic reticulum stress sensor activating transcription factor-6 leading to activation of the NLRP-3 inflammasome. J Biol Chem, 290(9), 5926-5939. doi:10.1074/jbc.M114.610899

Sokolowska, O., & Nowis, D. (2017). STING Signaling in Cancer Cells: Important or Not? Arch Immunol Ther Exp (Warsz). doi:10.1007/s00005-017-0481-7

Szebeni, J., Alving, C. R., Rosivall, L., Bunger, R., Baranyi, L., Bedocs, P., . . . Barenholz, Y. (2007). Animal models of complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles. J Liposome Res, 17(2), 107-117. doi:10.1080/08982100701375118

Toyama, T., Matsuda, H., Ishida, I., Tani, M., Kitaba, S., Sano, S., & Katayama, I. (2008). A case of toxic epidermal necrolysis-like dermatitis evolving from contact dermatitis of the hands associated with exposure to dendrimers. Contact Dermatitis, 59(2), 122-123. doi:10.1111/j.1600-0536.2008.01340.x

Vance, M. E., Kuiken, T., Vejerano, E. P., McGinnis, S. P., Hochella, M. F., Jr., Rejeski, D., & Hull, M. S. (2015). Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein J Nanotechnol, 6, 1769-1780. doi:10.3762/bjnano.6.181

Winter, M., Beer, H. D., Hornung, V., Kramer, U., Schins, R. P., & Forster, I. (2011). Activation of the inflammasome by amorphous silica and TiO2 nanoparticles in murine dendritic cells. Nanotoxicology, 5(3), 326-340. doi:10.3109/17435390.2010.506957

Zahr, A. S., Davis, C. A., & Pishko, M. V. (2006). Macrophage uptake of core-shell nanoparticles surface modified with poly(ethylene glycol). Langmuir, 22(19), 8178-8185. doi:10.1021/la060951b