Once the reader accepts a standard definition of nanotechnology, such as that offered by The Royal Society and The Royal Academy of Engineering,1 and then reads my definition of skin delivery available elsewhere (see Two Definitions),2,3 and then realizes that micro- and nanoparticles accumulate in the furrows and ridges on the skin surface where they act as a reservoir, then they are ready to ask the question answered in this article: Do nano-particles penetrate human skin?
Nanoparticles have been defined as single particles with a diameter less than 100 nm,4 which includes titanium
dioxide in transparent, inorganic sun care products, and is usually extended to 200 nm to include the zinc oxide in those products. But nanoparticles are only a subset of nanomaterials, which can also include cyclodextrins and liposomes. While cyclodextrins do not represent a nanotechnology in this author's opinion, liposomes do. However, liposomes will not be discussed in this article because unlike nanoparticles that are intended to rest on the skin, liposomes were specifically designed to penetrate the skin. Thus, the nanoparticles addressed in this article are solid particles with a diameter less than 200 nm.
Consider the following quote:
"In one of the most dramatic failures of regulation since the introduction of asbestos, corporations around the world are rapidly introducing thousands of tonnes of nanomaterials into the environment and onto the faces and hands of hundreds of millions of people, despite the growing body of evidence indicating that nanomaterials can be toxic for humans and the environment."
This is the first sentence of the executive summary of the May 2006 Friends of the Earth report, Nano-materials, sunscreens and cosmetics: small ingredients, big risks. 5 It is also found in the introduction and on the back cover of the same report. With such emphasis on this statement, one would think there is something fundamentally wrong with nanotechnology. The best way to analyze this is to study whether the "growing body of evidence" actually supports the claim that "nanomaterials can be toxic for humans and the environment."
Two things are essential for nanomaterials to constitute a risk, as suggested by The Friends of the Earth report. Humans need to be exposed to the nanoparticles and there needs to be an intrinsic safety hazard of these materials. Are humans indeed being exposed to tons of these materials by the various industries?
Apart from natural nanoparticles that occur in the environment—such as volcanic dust, lunar dust, magnetotactic bacteria and minimal composites—man-made industrial processes also create incidental nanoparticles such as diesel exhaust, coal combustion and welding fumes. A last group consists of the engineered nanoparticles that are created either top-down (via milling) or bottom-up (via crystal growth).6 Most nanoscale materials, whether engineered or natural, fall into one of four categories (although other ways of classifying them also exist6):
- Metal oxides, such as zinc and titanium oxide, that are used in ceramics, chemical polishing agents, scratch-resistant coatings, cosmetics and sunscreens;
- Nanoclays; naturally occurring plate-like clay particles that strengthen or harden materials or make them flame retardant;
- Nanotubes, which are used in coatings to dissipate or minimize static electricity (e.g., in fuel lines, in hard disk handling trays, or in automobile bodies to be painted electrostatically); and
- Quantum dots, used in exploratory medicine or in the self-assembly of nanoelectronic structures.
The cosmetic industry's contribution to the "thousands of tonnes of nano-materials" is mainly the global production of nanoparticles for sunscreen products, which was estimated to be approximately 1,000 tons during 2003-2004.7
As previously mentioned, the more flexible vesicles such as liposomes, elastosomes and other skin delivery systems are also an application of nanotechnology but their contribution to the production of "thousands of tonnes of nanomaterials" will be less--although they may have been produced over a longer period of time because liposomes were invented by Bangham8 in 1965 and have been used ever since.
The global production of engineered nanomaterials is estimated to increase from 1,100 tons in 2003-2004 to
5,700 tons in 2020, whereas the contribution of metal oxides in sunscreens is estimated to remain constant at
1,000 tons over the 2003-2020 time span investigated; the contribution of the cosmetic industry to the global production of nanomaterials will remain constant in absolute terms and reduce in relative terms.7
Therefore, nanomaterials are indeed present in the immediate environment, even in thousands of tons. But there is still a difference between the presence of nanomaterials in the environment and their getting under the skin of individuals (i.e., human exposure). The following discussion will thus focus on topical exposure and the skin penetration of nanomaterials that are used for cosmetic purposes, such as the metal oxides. Liposomes as well as other routes of exposure to nanomaterials, such as inhalation and oral uptake, will not be discussed.
Principle of Skin Penetration by Nanoparticles
Having established that there are indeed nanoparticles in the immediate environment, does this particulate matter penetrate human skin under normal application conditions? Let us zoom in on the principle of skin penetration of nanoparticles.
For ideal skin penetration, the penetrating molecule should have an octanol/water partition coefficient, K, of 10-100 (or 10log K = 1-2); its molecular weight should be below 500 Dalton; the molecule should be nonionized in the pH range (4.7-7.4) it will encounter during its transport through the skin; the molecule should have a high dipole moment and it should be a liquid at physiological temperatures.9 Particles, on the other hand, consist of many molecules and will therefore have a molecular weight that is much greater than 500 Dalton. They are also, by definition, not solubilized. Particles therefore do not have the optimal characteristics to penetrate human skin.
Professor Mike Roberts of the University of Queensland, Australia, calculated the exposure levels of nano-sized materials on purely theoretical grounds (see Figure 1).10 He argued the following:
1. Basic pharmacokinetics dictates that the steady-state epidermal exposure concentration of a compound is equal to the maximum flux of this compound divided by the sum of the desquamation clearance, the epidermal clearance and the return penetration (see Reference 2 for details).
2. The maximum flux of a compound can be predicted (for solubilized molecules) from the molecular weight of the compound.11 Roberts modified this relationship to predict the same from the molar volume.
3. Making the assumption that this prediction can be extrapolated from small soluble molecules to bigger solid particles, the maximum flux of a particle can be predicted from the molar volume (MV) using the equation:
log Jmax ≈ 3.978 - 5.282 MV.
4. The desquamation rate of the stratum corneum was set at 14 days, meaning that in 14 days the stratum corneum is completely renewed.
5. The return penetration is assumed to be zero, which is in line with experimental findings in the scientific literature.
6. The epidermal clearance was calculated from the flux through the stratum corneum, which can be calculated from the permeability coefficient in equations such as the ones developed by Potts and Guy, Barratt, Mitragotri, etc., that can be calculated from equations that predict the stratum corneum permeability co-
7. When calculating the steady-state epidermal concentrations, it turned out that desquamation had a profound impact on the epidermal concentrations reached. If a safety margin of 100 was applied, the predicted epidermal levels of a solubilized molecule with a molecular weight of 800 would be approximately 10 nmol/mL, whereas that of a particle with a diameter of
30 nm would be 10-18 nmol/mL, which is a factor of 1019 smaller. (See Some Perspective on 10-18 nmol/mL.)
From this, it can be concluded that—theoretically&mdashthere will be no significant penetration of particulate matter into the viable epidermis, and hence the exposure should be very, very small. However, the Friends of the Earth would argue these are nothing but theoretical predictions and that real experimental data is necessary. And they would be fully right. Therefore, the next topic to be discussed is the "growing body of (experimental) evidence."
This body of evidence is indeed growing and it is growing rapidly. Most articles describing skin penetration of nanoparticles deal with particulate sunscreen agents such as titanium dioxide and zinc oxide.
Experimental Skin Penetration Data on Nanoparticles in Cosmetics
The first thing that becomes apparent when examining articles describing skin penetration of nanomaterials is that the transfollicular route of skin penetration plays an important role. This is not amazing considering that in 1993, Rolland et al.13 had already discussed how fluorescent particles with a diameter between 3 µm and 10 µm penetrated almost exclusively via the sebaceous glands and the transfollicular orifice, whereas particles smaller than 3 µm penetrated generally into the skin and those greater than 10 µm did not penetrate the stratum corneum at all.
Subsequent investigations conducted mainly by Jürgen Lademann and his team at the Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Charité-Universitätsmedizin Berlin, have shown that fluorescein that was covalently bound to microparticles penetrated more efficiently into the hair follicle than free fluorescein (see Figure 2).14 In the stratum corneum, the topically applied substances were stored only in the upper layers of the corneocytes. These layers become a short-term reservoir because, every day, one layer is sloughed off. In contrast, the orifices around the follicles represent a long-term reservoir because their depletion only takes place by hair growth or sebum production, which are slow processes.14
This long-term reservoir function of the infundibulum, the orifice in the skin where a hair follicle emerges from the skin surface, was beautifully visualized in an article by the same group (see Figure 3).15 It was shown that the smaller microparticles penetrate deeper into the stratum corneum, especially in combination with cyanoacrylate skin stripping.16
At a given depth, a higher percentage of hair follicles showed penetration of microspheres when the diameter was 0.75 and 1.5 µm than when their diameter was 3.0 and 6.0 µm, although these larger microparticles were still small enough to penetrate, in line with earlier findings.13 But even for the smallest microspheres tested, the maximum percentage of hair follicles showing penetration was only 60%, even at very superficial depths (less than 200 µm).16 This suggested that a large fraction of hair follicles do not show any skin penetration.
The reason why so many hair follicles do not show any skin penetration is that not all hair follicles are "available" for uptake of particles. The "availability" for transfollicular penetration depends on the conditions of application.17 If massage is applied during the application, the hair follicles open up and the skin penetration from particle-containing emulsions is statistically significantly enhanced (i.e., deeper). The average penetration is around 1500 µm relative to non-particle-containing emulsions where penetration averages around 500 µm. This is similar to what was shown in Figure 2, a picture that was also obtained following application with massage. When the same formulations were applied without massage, the average penetration was less deep and around 300 µm, irrespective of using particle-containing emulsions or non-particle-containing emulsions.17, 18
What is happening during massage? Massage actually opens a closed hair follicle. Follicles can be closed due to a cover of shed corneocytes that act as a plug. Follicles normally open due to sebum flow and hair growth, as demonstrated by the finding that dye penetrates whenever sebum secretion and/or hair growth is occurring.18 Investigations revealed that 74% of hair follicles on the upper forearm were open but that processes such as massaging and chemical peeling increased this percentage to 100%.19 Follicles can also be experimentally closed by blocking them with a nail varnish.20
The enhanced follicular penetration of nanomaterials via open follicles is speculated to be caused by the movement of the hair follicle (mimicked by massage) relative to the nanoparticle. Topically applied particles may be entrapped under the cuticular cells of the hair shaft and may be guided further down along the hair follicle duct as the hair moves back and forth. This concept of particle penetration into the hair follicles has recently been introduced by the Lademann group and termed the geared pump hypothesis.18
Experimental Data on Penetration to the Viable Epidermis
Theory dictates that particles do not penetrate to the viable epidermis, but what do the experiments show? Particles, especially the smaller ones, do penetrate into the infundibulum, although to pull them in, massage is required. Is this creating an exposure as described above, or do the nanoparticles merely constitute a reservoir without any further penetration into the living tissues, as suggested in Figure 3?
Deeper penetration into the viable layers of the epidermis was recently investigated by Nohynek et al. who reviewed every available scientific article that describes the skin penetration of titanium and zinc oxide nano-particles.21 The articles that were published between 1996 and 2007 describe the skin penetration of titanium dioxide (with various types of coating) and zinc oxide (uncoated or no information available) of various particle sizes ranging from ultrafine 14 nm to 2 µm with the majority in the 10-100 nm range, using a variety of techniques (mainly in vitro skin penetration but some skin biopsies and skin stripping) on various species (human, rabbit, pig, mouse) on predominantly healthy and occasionally psoriatic skin. While these conditions varied widely, the results were remarkably consistent. "Penetration of particles into the stratum corneum and outer hair follicle; no penetration into living skin" or something similar was concluded roughly 16 out of 20 times.21
Before reaching a general conclusion that titanium dioxide and zinc oxide nanoparticles do not penetrate beyond the stratum corneum, it is necessary to have a closer look at the four papers that reached a slightly different conclusion.
The first is a study of Pirot et al. using zinc oxide (no information on particle coating) of unknown (microfine) particle size on human skin in vitro. Here, 0.34% was absorbed (location not specified) in 72 hr.22
The second study used zinc oxide of various non-specified particle sizes on normal and psoriatic human subjects, where zinc levels were measured after topical application and in vitro on pig skin. No increase in plasma levels was detected in vivo but in the in vitro experiments, skin penetration was less than 1% of the applied dose, whereas most zinc oxide was recovered in the stratum corneum.23
The third study involved again zinc oxide particles, this time of 15-30 nm, using human skin in vitro. Less than 0.03% of the applied zinc was recovered in the receptor solution, whereas no particles were detected in the epidermis or the dermis.24
The fourth study used titanium dioxide coated with dimethicone or silicone dioxide, 30-60 nm, and zinc oxide (uncoated, < 160 nm) in combination with in vitro pig skin and found no penetration beyond the stratum corneum, yet recoveries in the receptor fluid were 0.8-1.4% of the applied dose.25
When discussing these papers, Nohynek et al.21 state the following: "Similarly, as shown in recent in vitro percutaneous penetration studies, ZnO nanoparticles showed negligible penetration into pig25 and human skin.24 These findings confirmed the results of a number of in vitro or in vivo percutaneous penetration studies on ZnO particles that were reviewed in the opinion from the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP).23 None of these studies suggested significant penetration into or through living human or animal skin."
The reason for the increased zinc levels in the third paper, for instance, was the slight dissolution of the zinc oxide particles with subsequent penetration of the zinc ion.24 Because the first and second studies also measured zinc ion levels, this may also have been the case in the studies of the third paper, and therefore not be evidence of zinc oxide nanoparticle penetration. Nohynek et al. thus concluded that "most available theoretical and experimental evidence suggests that insoluble nanoparticles do not penetrate into or through normal as well as compromised human skin,"21--a conclusion with which this author agrees.
Are there therefore no concerns whatsoever regarding topical application of nano-sized components? The only papers describing skin penetration of nanomaterials into the living epidermis are from Nancy Monteiro-Riviere's group, which in 2006 suggested that quantum dots may penetrate into the epidermis or dermis of intact porcine skin.26 Quantum dots are nanocrystals that are used for imaging purposes in medical diagnostics and not in cosmetics. An extended discussion of Monteiro-Riviere's work26,27 on skin penetration of quantum dots is available elsewhere.28
Even if quantum dots do penetrate skin, what does this mean for the skin penetration of nanomaterials applied in cosmetic products? Reading and summarizing this "growing body of (experimental) evidence" already cited10,17,18,26,27 it can be concluded that the systemic exposure of humans following topical application to nanomaterials is very low but not necessarily zero. The highest skin penetration of nanoparticles can be achieved when nanoparticles with particle sizes below 10 nm are used under massage (or flexing) on rodent skin under in vitro conditions.
In cosmetics, nanomaterials are predominantly found in sun care products where the optimum particle size for high UVB and UVA attenuation but good transparency in the visible region is between 40 and 60 nm (see Figure 4).29 Such particles are much bigger than the quantum dots of which the skin penetration was described above. Sun care products are normally applied under massage but on human skin in vivo where the hair density is comparable to that on pig skin (average 20 (11-25) hairs/cm2 for porcine skin30 versus 14-32 vellus hairs on human skin).31
Apart from being applied with massage, all other requirements for optimal skin penetration of nanoparticles from sun care products are not met. Based on the "growing body of (experimental) evidence," it is highly unlikely that nanoparticles like titanium dioxide and zinc oxide as used in sun care products will penetrate into the living epidermis.
The Intrinsic Hazard of Nanomaterials in the Living Epidermis
If penetration does in fact occur, what is known about the intrinsic hazard of these nanomaterials? If some nanomaterial is reaching the living epidermis, irrespective of how little, the next issue to address is their intrinsic hazard. Do the few nanoparticles that manage to get into the viable epidermis constitute a health hazard?
Not being a toxicologist, this author must refrain from making conclusive statements on the intrinsic safety of nanomaterials in general, but for sun care, one can rely on the opinions made by the Scientific Committee on Consumer Products (SCCP) and other papers available in the scientific literature. Some recent reviews cited here refer to many other papers that describe the intrinsic safety (i.e., the hazard) of nanomaterials.
A 2007 review concluded that studies on wear debris particles from surgical implants and other toxicity studies on insoluble particles support the traditional toxicology view that the hazard of small particles is mainly defined by the intrinsic toxicology of particles (i.e., their chemistry), as distinct from their particle size.21
An updated version of the same review was published in June 2008 and reached the same conclusion: "Overall, the current evidence suggests that nano-sized cosmetic or sunscreen ingredients pose no potential risk to human health, whereas their use in sunscreens has large benefits, such as the protection of human skin against skin cancer."32
A 2006 review concluded that "there is currently little evidence from skin penetration studies that dermal applications of metal oxide nanoparticles used in sunscreens lead to systemic exposure. However, the question has been raised whether the usual testing with healthy, intact skin will be sufficient."7
In a review by independent science and technology journalist Trudy Bell, former editor for Scientific American, wrote that size matters, but so do shape and purity. She cautioned that general statements cannot be made presently. She also urged researchers to:6
- consider the original sources;
- look for appropriate qualifiers (such as preliminary);
- look for issues of scale (Is this substance also toxic in different forms, or in solution?);
- check whether reported exposures were actually to nanomaterials rather than micrometer-sized particles and to individual nanomaterials;
- be cautious about generalizing results from one study to another;
- not assume that experimental results can be extended to actual biological systems or to the environment;
- probe possible other reasons for toxicity; and
- not assume that common-sense macroscopic physics holds at the nanoscale.
She also stated that good R&D takes time. It is therefore not surprising that she does not claim a position on the safety of nanotechnology or nanomaterials. It would be wise to consider her cautionary warnings when reading reports that do make such claims.5, 7, 21, 29 Actually, Nohynek et al. already do this when they state that in vitro cytotoxicity, genotoxicity and phenogenotoxicity studies on titanium dioxide or other insoluble nanoparticles reporting uptake by cells, oxidative cell damage, or genotoxicity should be interpreted with caution since such toxicities may be secondary to phagocytosis of mammalian cells exposed to high concentrations of insoluble particles.21
This author therefore agrees with their summary conclusion that "Overall, the current weight of evidence suggests that nanomaterials such as nano-sized vesicles or TiO2 and ZnO nanoparticles currently used in cosmetic preparations or sunscreens pose no risk to human skin or human health, although other nanoparticles may have properties that warrant safety evaluation on a case-by-case basis before human use."
Nanotechnology: Infinitely Small or Infinite Possibilities?
After having established that human exposure to nanomaterials used in cosmetics is infinitely small, and that the hazards are over-estimated and more caused by the experimental conditions than reflecting what might occur in real life, it can be concluded that the risk of nanotechnology in cosmetics via topical application is also over-estimated. But what are the benefits of using nano-sized materials in general and in cosmetics in particular?
The unique size-dependent properties of nanomaterials mean that in some ways they behave like new chemical substances. For example, nanoparticles can scatter and absorb short-wavelength UV radiation but leave longer-wavelength visible light virtually unaffected (see Figure 4). Quantum dots can be used in medical imaging techniques because when they absorb UV radiation, they emit visible light, and the color of the emitted light differs for nanoparticles of different diameters.7 But there are also some very specific purely cosmetic benefits, other than translucent sun care products, that can be obtained from the use of nanoparticles in cosmetics.
Souto and Müller describe the cosmetic features of two nanostructured delivery systems: solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs).33, 34 They mention both the skin protective and lubrication/emolliency properties of these nanoparticles. When applying lipid particles onto the skin, a film layer will be formed, having a surface area that depends on particle size. The air-filled space within a layer of optimal packing density is independent of the particle size, which is considered to be 24% if assuming a three-dimensional hexagonal packing of ideal spherical-like particles. However, the air channels in a layer of nanoparticles (see Figure 5; top) will be much smaller than the air channels in a layer of microparticles (see Figure 5; bottom). The transepidermal water loss will therefore be more reduced in a formulation containing nanoparticles than in a formulation containing microparticles.34 Experimentally, this was also shown to be the case. The occlusion factor of lipid micro-particles with a diameter greater than1 m was only 10%, relative to a factor of 50% when using lipid nanoparticles of approximately 200 nm.35
Souto and Müller also claim that this occlusion may help to enhance the skin elasticity and skin penetration of active ingredients, but this depends first of all on the polarity of the active ingredient and there are many alternatives to nanoparticles available to increase the skin elasticity or moisture content of the skin. The same applies for their reputed lubricating effects that are due to their spherical-like shape.34 But these cosmetic benefits of nanoparticles are indeed only secondary because there are many other ingredients offering the same effect.
In this article titled "Nanotechnology and Skin Delivery: Infinitely Small or Infinite Possibilities?" it was concluded that the penetration of the particulate matter is infinitely small indeed. Nano-sized structures that are flexible will go into the skin, as shown in Figure 6 and exemplified by liposomes, which were designed to penetrate the skin. The interested reader is directed to References 28 and 36-38 for more on the skin penetration, hazards and biocompatibility of liposomes.
Finally, to achieve penetration the formulation must be massaged into the skin to open the orifice surrounding the hair follicles. However, the nanomaterials used in cosmetics such as the metal oxides are too rigid to penetrate, too big to penetrate, and are effectively cleared away from the skin by sebum flow, hair growth and desquamation, and we humans are simply not hairy enough to allow them to penetrate.
This suggests endless opportunities for nanotechnology in cosmetics, but this is also not true. The only reasons that particulate systems are used are purely cosmetic. Customers prefer transparent sunscreen formulations and for that, nano-sized particles are required. The skin moisturization and elasticity and lubrication benefits offered by the lipid nanomaterials are really secondary benefits, for which many other cosmetic ingredients are available that could offer more effect for less cost.
Therefore, it can be concluded that both the risks and the benefits of nanotechnology in cosmetics are exaggerated but it remains strange that there is a perceived risk for those topically applied nanoparticles such as the metal oxides for which there is no evidence of skin penetration, whereas there is no perceived risk for those topically applied nanomaterials such as elastosomes and flexible liposomes for which there is evidence of skin penetration. The latter is correct; the former may hopefully have changed after reading all these pages.
Despite this cautionary note, the future of nanotechnology in cosmetics is bright and shiny. This author foresees the main future benefits of nanotechnology to come from the combination of nano-sized materials and trans-follicular delivery, another topic soon to be discussed in Cosmetics & Toiletries magazine. Frum, for instance, concluded in 2007 that this route of penetration may account for up to 60% of all skin penetration of soluble materials.39 Combine this potential with the capability of nanomaterials to accumulate in the transfollicular orifice that acts as a long-term reservoir, and the following options will become a reality.
First, include solutes into SLNs and NLCs that accumulate in the orifice and subsequently diffuse out of these nano-sized delivery systems and provide a long-term delivery. Second, nano-sized solids with some minimal fat solubility will automatically accumulate in the orifice and then be delivered. Finding the right solubilities will be the challenge to get this to work but when successful, delivery from nano-sized particles will become gigabig.
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1. The Royal Society and The Royal Academy of Engineering, Nanoscience and nanotechnologies: Opportunities and uncertainties, The Royal Society and The Royal Academy of Engineering, London, UK, 2004, available at: www.nanotec.org.uk/report/summary.pdf (Accessed on Sep 21, 2008)
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