interactions of carbon nanotubes and fullerenes with the immune system of the skin and the possible implications related to cutaneous nanotoxicity

the understanding of the interaction of carbon nanotubes and fullerenes with the constituents of the skin, especially the skin immune unit, is relevant to the determination of toxicological endpoints. A systematic review was done focused on such aspects. Considerable part of the found references concentrated in cytotoxicity and skin permeation. On a smaller scale, there are articles on immunomodulation and activation of immune cells and other elements. Few of the found studies deal specifically with cutaneous immune response, limiting the related knowledge. The findings suggest that nanomaterials studied may be involved in skin problems such irritant contact dermatitis, anaphylactoid reactions, urticaria, angioedema, and raised the need for performing additional studies to confirm the findings. The standardization of the description and testing of nanomaterials characteristics used in experiments can facilitate comparison of results.


Introduction
Nanoscience has been developing at a fast pace, and nanomaterials can now be found in a wide range of products such as electronics, food, pharmaceutical products, and industrial and domestic utensils.
For a material to be considered as a product of nanotechnology, at least one of its dimensions must be between 1 and 100 nm 1 .This nanometric size means that most atoms in the molecule are on its surface, which explains the high reactivity of nanomaterials and their various applications 2 .
Among nanomaterials, the production of carbon allotropes such as fullerenes and carbon nanotubes (CNts) is the highest, and they have the most applications 3 .therefore, there is an increasing concern about risks associated with the environment and human health because of occupational exposure to these nanomaterials, in particular via the skin and airways 4,5 .the skin is a major barrier between man and the environment, and it plays an important role in the protection against physical, chemical, and biological aggressions 6,7 .Exposure of the skin to nanomaterials can occur via direct application of medications that contain nanocarriers, through tissues treated with nanoparticles, or involuntarily via contact with raw materials or residues from industrial production processes 8 .there are several issues regarding the interaction of nanomaterials with the skin.these include penetration through intact and damaged physiological barriers, cytotoxicity, induction of oxidative stress and inflammation, and mutagenicity 9 .
Similar to any new chemical substance, nanomaterials pose potential risks of allergic or irritant contact dermatitis because these structures can represent new allergens, haptens, and cross-reaction agents and cause unknown damages 10 .thus, many of the predicted risks associated with the interaction of nanomaterials with the skin involve the skin's immune response elements.it is important to understand each phase of this process to acquire knowledge about skin nanotoxicity.

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is not only to search for new applications of these nanomaterials but also to predict possible complications related to a prolonged stimulus of the immune response.

SIS
the skin is constantly exposed to numerous antigens and irritants, leading to the need for an effective and differentiated immune system.the diversity of the skin's immune response has led to the hypothesis, in the 20 th century, about the existence of a specific lymphoid tissue associated with the skin, the skin-associated lymphoid tissue (SAlt) 6 , which was later termed SiS 39 .
With regard to the type of immune response, the immune system of the skin includes elements of innate and adaptive immune response in addition to elements that mediate the two types of responses.

The skin physical barrier
the skin is composed of three layers, namely the epidermis, dermis, and hypodermis, which differ structurally and functionally 40 and together represent a physical barrier.the cornea layer is composed of corneocytes 41 that are connected to each other through corneodesmosomes and are surrounded by an extracellular matrix rich in nonpolar lipids, organized in a double layer.In the epidermis, keratinocytes are covered by the cornea and are organized in a manner that prevents the entry of microorganisms and potentially harmful chemical substances 42 .They are the predominant cells organized in four layers (basal, spinosum, granulosum, and stratum corneum) and are connected to each other through desmosomes and to the basal layer through hemidesmosomes.these cells are not inert and participate in the immune response by secreting cytokines, chemokines, antimicrobial peptides, components of the complement system, and arachidonic acid metabolites 43,44,45 . in addition, they are involved in the phagocytosis of bacteria and fungi 44 .langerhans cells (LCs) and Merckel cells and a fluctuating population of inflammatory cells lie between keratinocytes and melanocytes 40 .
Underneath the epidermis is the basal membrane, followed by the dermis, with a diversity of cells such as fibroblasts, mastocytes, macrophages, and transitory inflammatory cells as well as the skin appendixes and the vascular and nervous plexus 40,45 .the skin annexes consist of the pilosebaceous unit and the sweat glands.transcutaneous penetration of chemical products occurs via passive diffusion according to different concentration gradients 46 ; usually, only particles that weigh up to 500 Da can penetrate the skin 47 .In general, structures with a size of up to 1000 nm penetrate the intact skin in flexural areas; in other areas, if the skin is not intact, particles of up to 7,000 nm can reach the deeper skin strata 10 .this process presents physical obstacles such as the corneal layer, an essentially lipophilic structure 48 .
Sweat glands and hair follicles represent points that exhibit lower resistance to the penetration of substances into the skin because they have direct openings on the skin surface 48 . in general, it has been reported that particles smaller than the diameter of the follicular opening, between 10 and 210 µm, can penetrate the skin through that route 48 .

Cells involved in the skin's innate response
Neutrophils are the major components of the innate immune response, although they are not naturally present in the skin 48 .
Monocytes leave the blood circulation and reach the skin, where they transform into macrophages 49 ; they exist in large numbers, particularly in the dermis, near the basal membrane 50,51 .
Antigen-presenting cells are responsible for the internalization, processing, and presenting of antigens to t cells in lymph nodes, thereby contributing to acquired immunity 52 . in the skin, two populations of these cells are observed: lCs and dermal dendritic cells 53 .lCs are a subtype of immature dendritic cells present in the epidermis 54 , in the basal and suprabasal layers, whose extensions cover an area of approximately 20% of the epidermis, thereby facilitating antigen capture 55 .Dendritic cells are predominantly found in the dermis and perform functions to those of lCs 53 .
Mastocytes present in the skin are located around capillaries, arterioles, and vennules of the dermis and hypodermis 50 .
Basophils are present in lesions in some skin disorders such as atopic dermatitis, hives, and itching, and they are structurally and functionally similar to mastocytes 56 .the distribution pattern of eosinophils in the skin is similar to that in other tissues; their number increases during allergic and autoimmune processes 54,57,58 .

Soluble elements of the skin innate immune response
Proteins of the complement system reach the skin via diffusion through vessels that have their permeability altered by an inflammatory stimulus 50 .Other cells such as monocytes and macrophages present in the skin or attracted to it at the start of inflammation can also be a source of these proteins.The synthesis of several elements of the complement system by keratinocytes, such as C3; C5; C7; C8; C9; factor B; regulatory factor H; factor i; complement receptors Cr1, cC1qr, C5ar, and Cr 2; and regulatory cytokines has been described previously 59 .

Cells involved in the skin adaptive response: T and B lymphocytes
t lymphocytes represent 1% to 3% of the population of epidermal cells 39,50,60 , whereas lymphocytes B are not found in the intact skin or oral mucosa 39 .these t cells are memory CD45r0+ lymphocytes that express adhesion molecules on their surface, such as the cutaneous lymphocyte antigen (ClA), in addition to chemokine receptors and enzymes that contribute to migration to the skin 61 .
Under homeostasis conditions, t lymphocytes are present in the epidermis near the basal membrane and lCs and in the dermoepidermal junction around the postcapillary vennules of the superficial and deep plexus 50,39 .

Method
A bibliographic review of scientific articles was conducted, including clinical and laboratory studies, case reports, reviews, editorials, letters, and websites on the topic.the bibliographic search focused on the immune system, anatomy, and specificities of skin immunology, carbon allotropes, and their interactions.We considered articles and documents (printed or online) published until November 30, 2011.the electronic databases Academic Search Premier (EBSCO), Cross Search (isi Web Services WOK), Medline, Ovid MEDliNE, Scielo, and Scopus were used for the bibliographic search.Bibliographies of the selected articles were also reviewed to search for unselected sources.the search was limited to documents written in English, French, Spanish, and Portuguese.
A list of the relevant applications and physical and chemical characteristics of the nanomaterials under study was prepared in addition to a brief description of the cutaneous immunological structure.Moreover, a report of the possible interactions of nanomaterials with each element of the skin immune response was prepared.

Interaction with the elements of the physical barrier and skin penetration
the penetration of CNts and fullerenes into keratinocytes and other skin cells has been studied not only to assess the toxic effects but also to develop matrices that carry pharmaceutical compounds.Monteiro-riviere et al. 62 demonstrated that MWCNts can penetrate cultured keratinocytes after observing them in cytoplasmic vacuoles via electron transmission microscopy (EtM).Bullard-Dillard et al. 63 described the ability of fullerene C 60 and of a more soluble version of the nanomaterial, fullerene C 60 , derived from quaternary ammonia salts, to be taken up by immortalized human keratinocytes.Both the fullerene forms were internalized by the keratinocytes, although the substance derived from ammonia salts accumulated more slowly.the precise mechanism of the penetration of carbon allotropes into keratinocytes has not been described, although this internalization probably occurs through endocytosis or phagocytosis 64 .
Analysis of cytotoxic effects of nanomaterials on cells of the cutaneous structure is relevant because their destruction can lead to alterations in the physical barrier as well as the production of cytokines that trigger the inflammatory process.
Shvedova et al. 25 assessed the effect of SWCNts in cultured Ha-Cat keratinocytes and observed the induction of free radicals synthesis, accumulation of peroxides derivatives, reduction of antioxidants, and subsequent toxic effects on keratinocytes.
Moreover, studies have suggested that regardless of the concentration of the nanomaterial in the medium, there is a difference in cytotoxicity among CNts in the following order: MWCNO < MWCNt < SWCNt 65,66 .this difference may be explained by the diameter: smaller structures may exhibit a higher cytotoxic activity.the cytotoxic effect of fullerenes on keratinocytes depends on the nature of the radical used in the functionalization of the nanomaterial and its concentration 24,67 .
Metal contaminants, present as residues of the process of nanomaterial synthesis, play a relevant role in cytotoxicity because they catalyze reactions that produce free radicals 25 .Murray et al. 26 exposed artificial human skin (EpidermFT) and murine epidermal cells JB6 P+ to partially purified and nonpurified SWCNTs containing iron. the JB6 P+ cells produced OH radicals when exposed to SWCNt; the process was inhibited when deferoxamine, a metal chelator, was added to the medium.The artificial skin, with human cells in the epidermis and dermis, after exposure to 75 µg of SWCNts, exhibited hyperkeratosis; parakeratosis; an increase in basal cells in the epidermis and in fibroblasts in the dermis; and an increase in collagen fibers.Moreover, there was an increase in the synthesis of inflammatory cytokines after exposure to nonpurified SWCNT, particularly IL-6, IL-12, and IFN-γ.
the association between the destruction of keratinocytes via the cytotoxic activity of the released cytokines and histological changes in the skin indicates a possible irritant contact dermatitis triggered by this class of nanomaterials 68 .
the interaction of nanomaterials with the immune system necessarily depends on the ability of these structures to penetrate the intact skin or skin damaged by trauma or inflammatory processes.This internalization can occur via hair follicles and sweat glands, between cells or through cells; in this case, it occurs via processes such as endocytosis, direct penetration of the cell membrane, or transport through transmembrane channels when the nanoparticle is smaller than 5 nm 50 .

Several characteristics intrinsic of nanomaterials appear
to be relevant for analysis of skin penetration, such as size, hydrodynamic diameter, surface charge, shape, composition, zeta potential, aggregation, metabolism, and deformity 50 .Furthermore, at present, there is a great concern regarding carbon allotropes and occupational exposure.Previous studies have simulated work conditions susceptible of altering this internationalization, such as performing repetitive movements.rouse et al. 69 have demonstrated the occurrence of passive diffusion of functionalized fullerenes [Baa-Lys (FITC)-NLS] in pig skin subjected to prolonged repetitive flections.ETM revealed that the penetration of these fullerenes predominantly occurred through the intercellular space and that after 24 h, they were located in the granular layer. in an attempt to simulate occupational exposure, Xia et al. 70

Interaction with neutrophils
Most studies on the interaction of carbon allotropes with neutrophils have focused on the airways.this is because exposure to CNts and fullerenes mostly occurs via the airways in occupational situations 71 (table 1).
Neutrophils internalize functionalized and nonfunctionalized carbon allotropes and accumulate them in intracytoplasmic vesicles.the surface charge of these structures appears to influence the action of neutrophilic enzymes, and SWCNTs with a negative surface charge can be degraded by myeloperoxidases present in cytoplasmic vesicles 72 .this fact is important because it suggests that changes in the surface of nanomaterials can alter the biopersistent nature of these structures, thereby increasing biocompatibility and reducing toxic effects when they are used, for example, as drug carriers.Kagan et al. 72 produced CNts after modeling sites active after the action of neutrophil myeloperoxidase.the results revealed that in contrast to compounds without the active sites under study, these compounds were susceptible to destruction by myeloperoxidase of laboratory animals, without the appearance of granulomatous lung lesions.
Once inside the cell, nanomaterials can affect the mechanisms of action of neutrophils.thus, it is possible that nanomaterials such as fullerenes, which have antioxidant action, neutralize the reactive oxygen species (ROS) of phagolysosomes.Because these oxygen species are important for the degranulation of neutrophils, the function may be reduced by the effect of the immune response to infectious agents.Moreover, this neutralization of ROS can be useful in treating inflammatory dermatosis such as acne 17 .
However, this depressor effect on the neutrophilic action was not observed in the study by tsao et al. 73 , where it was suggested that the carboxyfullerene increased the viability and bactericide power of these cells against gram-positive bacteria.
Moreover, other studies have demonstrated the pro-inflammatory action of carbon allotropes, particularly those associated with lung instillation of nanomaterials with increased neutrophilic infiltrate.Kagan et al. 72 have suggested that the anti-inflammatory effects observed for the allotropes occurred when the structures used in the experiments were not functionalized.
Additional studies need to be conducted for assessing the factors involved in these diverging responses.
A study with guinea pigs has suggested that skin contact with SWCNTs promotes the influx of neutrophils to the site only at high doses such as 160 μg 26 .the preferential location of the cell infiltrate around the follicles raises the question about a possible penetration via these structures.A study using CNts instilled in lungs has shown that the neutrophilic infiltrate only occurs in lung tissue if the nanomaterial is not aggregated 74 .

Interaction with macrophages
Phagocytosis of nanoparticles by macrophages depends on the physical and chemical properties of these structures, such as size, solubility, surface charge, and functionalization 75 .thus, larger nanoparticles with a surface charge would have a greater chance of being internalized via this process.
Opsonization of nanoparticles, with the adhesion of antibodies and fractions of the complement system to their surface, instantaneously occurs when they are administered parenterally, thereby boosting their phagocytosis 76 .this observation can be transposed to nanoparticles capable of entering the skin, which would be more easily internalized by macrophages once opsonized by antibodies and complement factors present in the epidermis and dermis.

Internalization of carbon allotropes by macrophages can
occur via phagocytosis, particularly when they are functionalized and aggregated 72,77 .Although most reports did not specify the term "pinocytosis," smaller and nonaggregated allotropes possibly penetrate the cell via this process.research on the cytotoxicity of carbon allotropes in macrophages is divergent.Although the reduction in cellular viability has not been demonstrated 77 , it has been reported that the toxicity of these nanomaterials is dependent on their concentration 67 , the presence of metal contaminants 24 , and their solubility 78 .the fact that different concentrations were used in the experiments may explain this difference.the study in which cytotoxicity was not observed used nanoparticles at a concentration of 10 μgMl −177 , whereas the other studies used concentrations ranging from 30 μgMl −1 to 0.5 mgMl −165, 67 .it has also been shown that for this cell line, there was a difference between the cytotoxic potential of SWCNts and MWCNts; therefore, the latter did not change cell viability even at high concentrations 67 .
With regard to functional changes, one study showed that only CNts capable of stimulating phagocytosis induced an increase in IL-6 and TNF-α, which was not observed for structures that enter the membrane of macrophages via a different process 77 .the function of macrophages with regard to usual stimuli, such as bacterial structures, can also be reduced after exposure to carbon allotropes.Dumortier et al. 77 have demonstrated that this fact depends on functionalization because only SWCNTs that contained polyethylene glycol (PEG) had an effect on the macrocytic function.
the induction of macrophage maturation by these nanomaterials, analyzed via the expression of surface molecules such as CD40 and CD86, showed divergent results.Although a lower concentration of SWCNts did not induce the expression of these molecules 77 , both macrophage maturation and activation were observed in the study in which a higher amount of nanomaterial was used 67 .thus, similar to the cytotoxic potential, macrophage activation by CNts and fullerenes may be dose-dependent.
The inability of macrophages to phagocytize inert materials such as nanomaterials has been observed even when they are opsonized 76 .this process may lead to the accumulation of monocytic cells around the nanomaterial, with the formation of granulomas 79 in addition to enzyme release by inflammatory cells such as neutrophils, which in turn may lead to tissue damage 76 . in a study in which the formation of skin gra-nulomas was analyzed, SWCNTs and MWCNTs of 20 and 80 nm, respectively, were implanted under the skin of guinea pigs 80 .the formation of granulomas was observed after 2 weeks; at a late stage, SWCNTs induced higher macrophage infiltration and the granulomas had thicker walls.The use of highly purified nanomaterials enabled the observation that these structures alone, without metal contaminants, can induce the formation of granulomas (table 1).

Interaction with dendritic cells
Analysis of the interaction of nanomaterials with dendritic cells, the most effective antigen-presenting cells of the human body, aims to study the possible internalization, processing, and presentation of nanomaterials, as if they were antigens.
Carbon allotropes can be internalized by dendritic cells via macropinocytosis 81 and phagocytosis 32,82 ; endocytosis has not been described in the reviewed studies.Internalization seems to be dependent on the concentration of the nanomaterial and on the size of the structures because those with a larger diameter are more easily internalized 82 .
the results of the cytotoxic potential of carbon allotropes are conflicting.One study has described that the toxic action of SWCNts and MWCNts in dendritic cells is dependent on the concentration of the nanomaterials (3, 10, 30 μgMl −1 ).
On the other hand, Wang et al. 82 have reported that carboxylic MWCNTs of different sizes and concentrations (10, 50, 100 μgMl −1 ), with metal contaminants, do not exhibit significant cytotoxic activity on these cells.In the first study, the physical and chemical characteristics of CNts were not described, such as functionalization, which could explain the differences that were observed.A higher concentration of the nanomaterial and the presence of varying amounts of metal contaminant would indicate a higher toxic effect, in contrast to what was observed by Wang et al. 82 .
Moreover, the potential immunomodulator role of carbon allotropes in dendritic cells has been studied.CNts can be conjugated with peptides considered as weak immunogens via unknown mechanisms and induce internalization by dendritic cells, thereby leading to the activation of CD4+ t lymphocytes, which in turn develop th2 immunity, with the production of antibodies 81 .this characteristic may have wide applicability in the development of immunotherapy, particularly in cancer treatment; tumor antigens that have been considered as weak immunogens till date may have their immune potential amplified by conjugation with nanomaterials.On the other hand, it remains unknown whether nanomaterials can bind to autologous peptides, turning them into strong immunogens, which are then recognized by antigen-presenting cells and result in the development of autoimmune diseases.
With regard to dendritic cell activation, which is an essential process for these cells to be able to present antigens to t cells, fullerenes and CNts exhibited distinct results.the former, according to Yang et al. 83 , can induce the expression of lymphocyte co-stimulatory molecules, chemokine receptors, and inflammatory cytokines on the surface of cells, whereas CNts the latter cannot 67,82 .the consequences of these processes should be studied.Usually, the presentation of antigens identified as potentially harmful to dendritic cells triggers their processing and cell maturation.thus, whether fullerene can be recognized as an antigen to be presented to T cells or whether it only interferes with cell-signaling mechanisms (with unknown effects) should be studied.in addition, the fact that CNts did not activate dendritic cells in the abovementioned experiments does not exclude the possibility that these nanomaterials act as haptens by binding peptides and becoming strong immunogens.
Although langerhans cells are important for the presentation of skin antigens, no studies analyzed this interaction using carbon allotropes.Skin dendritic cells were also not mentioned in the studies under analysis.the importance of reproducing the findings of studies using the human skin may be reinforced by other studies involving other nanomaterials applied in skin immunization.Manolova et al. 53 have demonstrated that skin antigen-presenting cells were only relevant for polystyrene internalization and transport when they were larger than 200 nm.Smaller structures could spontaneously migrate through skin lymphatic vessels to the skin's draining lymph nodes, where they would be internalized by dendritic cells.Thus, if carbon allotropes and silver nanoparticles exhibit a similar behavior, it is possible that they only interact with skin antigen-presenting cells if they are aggregated or if they are longer than 200 nm, in case of CNts (table 1).

Interaction with mastocytes
Carbon allotropes can be internalized by human skin mastocytes; a study using fullerenes showed that this process occurs via endocytosis 84 . in the cell, fullerenes can be found inside organelles and in the cytoplasm 84 .
Studies have indicated that carbon allotropes have distinct effects on mastocytes.Although CNts in high concentrations are associated with the influx of mastocytes to the application site and an increase in degranulation 26 , fullerenes appear to inhibit this process as well as release cytokines 85,86 .this fact may have implications in the use of these nanomaterials because CNts may be related to nonimmunological cases of hives and angioedema in addition to anaphylactoid reactions, whereas fullerenes may be employed to prevent these reactions.the location of fullerenes in lysosomes and the mitochondria, which are sites of rOS production, may explain the inhibitory action on mastocytes 84 (table 1).
Analysis of the effect of other factors in the activation of mastocytes, such as the concentration and functionalization of the nanomaterial, should be studied further.Even if the amount of histamine released is not sufficient to induce systemic alterations, it should be noted that the substances synthesized by mastocytes alter the vascular permeability, thereby favoring the migration of inflammatory cells to the skin and resulting in various effects.

Interaction with eosinophils
Although eosinophils participate in skin allergic processes, few studies have been found to focus on these cells and carbon allotropes.SWCNts, MWCNts, and carbon black injected in the airways of mice, associated with ovoalbumine allergen (OVA), can induce an increase in eosinophils in the absence of bronchoalveolar lavage 87 .However, an increase in eosinophils has not been observed in the experiments using skin implants of carbon allotropes (table 1).

Interaction with the complement system
Carbon allotropes can bind to some fractions of the complement system 29 and activate the classical route, alternative route 76 ,and lectin route 88 ; however, some results are conflicting 29 .this activation of the complement by CNts can be reduced or inhibited through changes in the surface of the nanomaterial, such as functionalizaton by covalent bonds 88 .the interaction of the proteins of the complement system with nanoparticles can increase the purification of these structures and thus reduce the bioavailability of nanoparticle drugs or induce a pseudoallergic and anaphylactoid reaction, the latter being caused by potent anaphylatoxins such as C4a, C3a, and C5a.
Despite the production of various elements of complement routes by cells of the epidermis and dermis and despite these factors reaching the skin through the bloodstream, no studies assessed the activation of the system at the site.

Interactions with T lymphocytes
Functionalized CNTs can penetrate T lymphocytes 77 , and studies on the cytotoxicity in these cells have discrepant results 77,89 , which may be explained by differences in the methods used in the experiments.Functionalization can increase cytotoxicity, which also depends on the time of exposure and on the quantity of CNt 89 .
With regard to lymphocyte function, CNts appear to favor the activation of CD4+ t lymphocytes over CD8+ t cells.Koyama et al. 80

Interaction with B lymphocytes and antibodies
CNts and fullerenes appear to be able to enter B lymphocytes 78,91 .Although functionalized SWCNTs do not activate B cells and are not toxic to them 77 , experiments with fullerenes have shown cytotoxicity and immunosuppression of B lymphocytes 90,91 . in this case, there should be some caution regarding variation in the concentration of the nanomaterial and the presence of functionalization.
Another interesting point in the study of B cells is their ability to recognize nanomaterials as antigens and trigger an undesired production of antibodies.Chen et al. 92 have demonstrated the development of igG antibodies against C 60 fullerenes, with cross--reaction with C 70 .This finding may be important for the development of vaccines and for the assessment of risks associated with the use of carbon allotropes in pharmaceutical products.the induction of igG directed against nanomaterials may be related to the reduction in the viability of the administered drugs as well as to inflammatory processes by the deposition of immunoglobulin.
The remaining antibodies in the organism, nonspecific for carbon allotropes, have a weak interaction with these nanomaterials and are thus not relevant to the opsonization process 92 (table 2).

Conclusion
the evolution of nanotechnology depends on the development of biocompatible nanomaterials, which involves understanding the interaction between these structures and the immune system.
In it is often difficult to distinguish between innate and adaptive immunity owing to the interconnection between these two types of immune responses.Although the isolated description of each component of immunity facilitates the assessment, it may hide the interaction between them.
the interaction between carbon allotropes and various elements of the innate and adaptive immune responses is wide and complex.Most cited studies have referred to experiments conducted in mice or in vitro; in the latter case, the cells of the immune system that were used were not necessarily human skin cells.thus, caution should be taken when transposing the observed results to the human skin because immune interactions in living organisms may alter the responses that are observed in in vitro experiments.
Despite the importance of the physical and chemical characteristics of nanomaterials used in the experiments for assessing biological properties, no standardization was observed in their description.this fact, in addition to the use of different techniques, may hinder data interpretation.in general, carbon allotropes are potentially involved in irritant and granulomatous skin reactions in addition to angioedema, hives, and anaphylactoid reactions.New studies are required to establish the risks and benefits resulting from the interaction between nanomaterials and SiS.
assessed the effect of different solvents on the penetration of fullerene C60-pristine in the skin of Yorkshire pigs.they observed that only fullerenes diluted in toluene, cyclohexane, and chloroform were present in the deep corneal layer.Although caution should be taken regarding the amounts and concentration of nanomaterials used in these experiments and their real concentration in the environment, one should be careful regarding the substances present in the workplace that may enhance the skin absorption and toxicity of nanomaterials.

table 2 .
implanted BAlB/c, SWCNts (0.8 to 2.2 nm), CSNts (cup stacked-type Nts) (50 to 150 nm), and MWCNts (20 and 80 nm) under the skin of mice and, in most experiments, observed an increase in CD4+ t lymphocytes in the peripheral blood and reduction in CD8+ t cells in the ini tial weeks (table2).Summary of the interactions between carbon allo-

table 1 .
Summary of the interactions between carbon allotropes and innate immune response cells the cytotoxicity observed in Jurkat t cells, commonly used in studies of t-cell viability, was not observed in human cells, which raises questions about the utility of this cell line.