Nanotechnology in dermatology
Published on: Mar 3, 2016
Transcripts - Nanotechnology in dermatology
9A. Nasir et al. (eds.), Nanotechnology in Dermatology,DOI 10.1007/978-1-4614-5034-4_2, © Springer Science+Business Media, LLC 20132.1 IntroductionExcessive exposure to ultraviolet (UV) radiationinduces a wide range of adverse effects such assunburn, photoaging, photoimmunosuppression,and photocarcinogenesis. Use of sunscreen is animportant practice by the public to protect againstexcessive UV exposure and reduce UV damages.In general, inorganic-based sunscreen composedof mineral UV ﬁlters, such as titanium dioxide(TiO2) and zinc oxide (ZnO), work by reﬂectingand scattering UV radiation. These agents areregarded as safe and effective. Compared toorganic UV ﬁlters, such as avobenzone and oxy-benzone, inorganic ﬁlters are less irritating onindividuals with sensitive skin and chronic skindisorders. For these reasons, TiO2and ZnO havebeen widely recommended as the safest UV ﬁltersin sunscreen products. Despite these beneﬁts,older sunscreens containing these ingredientswere limited in popularity by their poor cosmeticappearance. Due to the broad particle size distri-bution and poor dispersive qualities of the TiO2and ZnO particles, these sunscreens left a white oropaque ﬁlm, as well as grainy-residue on the skin.The diminished aesthetics of these sunscreenshindered wide acceptance by the public.This problem was met with a solution in nano-technology. Nanotechnology involves the design,production, and application of materials in thesize range of 1–100 nm. As existing materials arereduced to this size, a new set of physical, chemical,mechanical, and electrical properties are revealed.Application of this technology has aided in thedevelopment and advancement of new tools innumerous ﬁelds. Today, nanomaterials are foundin electronics, paints, foods, cosmetics, and coat-ings and are increasingly applied to the medicalﬁeld in diagnosis and drug delivery. The adventof nanotechnology also brought about greatconcern for the potential risks and toxicity ofthese foreign materials. Concerns surroundingnanotechnology in cosmetics and sunscreens pre-dominantly surround penetration into humanskin and possible systemic exposure from topicalapplication.2.2 History of Nanosized TiO2and ZnOThe broad distribution of particle size in older-generation sunscreens caused excessive whiteningof the skin, resulting in consumer reluctance to useproducts containing TiO2and ZnO. The solutionarose from reducing their size to nanoparticles.The average size of these minerals was <100 nm,L.L. Chen, B.A. • S.Q. Wang, M.D.(*)Dermatology Service, Memorial Sloan-KetteringCancer Center, 160 E 53rd Street, New York,NY 10022, USAe-mail: email@example.comI. Tooley, Ph.D.Croda Europe Ltd, Sun Care and Biotechnology,Foundry Lane, Ditton, Widnes, Cheshire WA8 8UB, UK2Nanotechnology in PhotoprotectionLucy L. Chen, Ian Tooley, and Steve Q. Wang
10 L.L. Chen et al.had superior UV protection, and had improvedcosmetic appearance. By the 1980s, patents wereﬁled and commercial sunscreens containing TiO2nanoparticles were introduced on a large scale by1990. Nanosized ZnO was used in the later part ofthe decade . Since that time, nanosized TiO2and ZnO ingredients have been approved anddeemed safe for use by various consortiums andorganizations including the US Food and DrugAdministration (FDA). These materials now rep-resent one of the largest applications of oxidenanoparticles with estimations that 70 % of tita-nium sunscreens and 30 % of zinc sunscreens areformulated with nanoingredients .The current sunscreens on the market are moreaesthetically acceptable and superior to older-generation sunscreens due to nanoparticle tech-nology. In terms of cosmetic elegance, the smallerparticle sizes minimize visible light scattering sothe resulting topical application appears “trans-parent.” However, it is a misconception that thenano- TiO2and ZnO particles are truly transpar-ent. In reality, they appear transparent at or belowconcentration thresholds due to increased lighttransmittance in the visible light range. Not onlymust particle size remain small, but also the par-ticle size distribution must be narrow to avoidany whitening effect. Mineral particles of TiO2reﬂect and scatter UV light most efﬁciently at as i z eof 30–100 nm; whereas ZnO has an optimal sizeof 60–100 nm particles [3, 4]. Another beneﬁt ofnanoingredients is the greater ease of the productto blend into the skin, due to the small particulatesize. The reduction in particle size scatters andreﬂects UV more efﬁciently, improving humanskin protection against UV-induced damage. Forall of these reasons, nanometal oxides have beenincluded into sunscreen products.2.3 Three States of Nano TiO2and ZnONanosized TiO2and ZnO can exist in three differ-ent states, encountered during the manufacturingprocess: primary nanoparticles (5–20 nm), aggre-gates (30–150 nm), and agglomerates (1–100 mm)(Fig. 2.1). The ﬁrst stage in manufacturing nano-particles is creation of the primary particles. Thestrong crystal attractions force primary particlesto cluster together, forming chemically boundaggregates. These aggregates represent the small-est units that actually occur in a ﬁnal sunscreenformulation because the forces required to breakapart aggregates are far greater than those encoun-tered during production of sunscreen formulationsor application onto the skin. Aggregates may alsoform loosely bound agglomerates with sizesgreater than 1 mm (1000 nm). This is the typicalsize of nano- TiO2and ZnO powder and resultsfrom the drying and heat treatment processes ofmanufacturing. The sunscreen’s vehicle formula-tion can also cause formation of large agglomeratesFig. 2.1 Formation of aggregates and agglomerates from nanoparticle building blocks
112 Nanotechnology in Photoprotectionwhich do not provide effective UV attenuation.Special inert coating materials (commonly silicaor dimethicone) can be applied to the surface ofmineral particles to improve dispersion in sun-screen products and resist the formation of largeparticle agglomerations . Nanoparticle-containing sunscreen emulsions can be visualizedunder transmission electron microscopy (TEM)(Fig. 2.2a, b). Typically, individual TiO2and ZnOnanoparticles cannot be detected, but presentmainly in clusters (usually aggregates of30–150 nm in size). On the market, sunscreengrade nanosized TiO2ranges from an ultra ﬁnesize of 30 nm to micro-sized aggregates. On theother hand, ZnO particles are used in the sizerange between 60 and 200 nm and the aggregateform is the smallest size that will occur in a sun-screen formulation.2.4 UV Attenuation Basedon Particle SizeNanosized ultraﬁne inorganic sunscreens havethe advantage of having both UV-reﬂecting andUV-absorbing properties. UV attenuation is thesum of scattering and absorption and depends onseveral factors including (1) dispersion of theinorganic particles (2) particle size and (3) refrac-tive index . With decreasing particle size, UVprotection shifts towards protection to shorterUV wavelengths. In other words, smaller particlesattenuate shorter wavelengths of UV radiation.By TEM studies of UV attenuation and particlesize, it is also known that nanoparticles reﬂectand absorb UV light most efﬁciently at the aggre-gate size (Fig. 2.3). At the average aggregate sizeof approximately 100 nm (red curve), TiO2par-ticles offer effective UVA and UVB coverage.However, signiﬁcant scattering is noted in thevisible region. In comparison, TiO2particles withan average aggregate size of approximately50 nm (green curve) have less visible light scat-tering and offer higher UVB and lower UVA pro-tection. TiO2particles with an average aggregatesize of 20 nm (blue curve) offer signiﬁcantlylower protection from UVA and UVB radiationcompared to 50- and 100-nm aggregates. Thesunscreen with a smaller particle size will be amore transparent product; however, its UVefﬁcacy will require additional UVA ﬁlters toachieve broad spectrum protection. Thereby, thebest sunscreen would be one containing a mix-ture of TiO2nanoparticle aggregate sizes rangingbetween 50 and 120 nm . ZnO has an optimalsize between 60 and 100 nm particles. To achievethe optimal size and desired UV absorptionproﬁle, nanometal oxides are usually stabilizedwith dispersing agents to maintain stability andprevent the reformation of agglomerates.Therefore, sunscreen formulators must ﬁnd anoptimum particle size in order to achieve thedesired UV absorption proﬁle while delivering apleasing aesthetic formula.Fig. 2.2 (a) 15 nm TiO2and (b) 35 nm TiO2primary particles and aggregates measured by TEM
12 L.L. Chen et al.2.5 Safety Concerns for Nano TiO2and ZnOWith the advent of TiO2and ZnO-based sun-screens, the safety concerns are centered aroundseveral issues: the endogenous toxicity of nano-particles, the potential toxicity on the epidermis,and the percutaneous penetration of nanomateri-als through skin. In their small size, nanoparticlesare able to form protein complexes, evade immu-nologic defense mechanisms, and most impor-tantly, induce free radical formation . Becauseof the small size of nanoparticles and theirincreased surface reactivity, there is a greaterpotential to cause harm than larger particles. Forexample, the large surface interface can result innanoparticles reacting with biologic proteins.When nanoparticles bind to proteins, they formcomplexes which can result in protein surfacemodiﬁcations and downstream signaling path-ways and metabolic routes which are differentfrom the original protein. In addition, nanoparti-cles can evade the human defense system byescaping phagocytosis, either due to their smallsize or by carrying unrecognizable surface pro-tein signals. Nanoingredients may also be inten-tionally engineered to avoid clearance in order toachieve therapeutic effects resulting in a pro-longed half-life, ability to penetrate the bloodbrain barrier, and preference to collect in speciﬁcorgans.Besides the endogenous toxicities of nanopar-ticles, there is concern that TiO2and ZnO in topi-cal sunscreens generate free radicals duringexposure to UV radiation. When exposed to UVradiation, TiO2and ZnO generate electrons in thelocal media which in turn can induce the formationof peroxides, free radicals, and other reactive oxygenspecies (ROS). In turn, these ROS can causedamage to proteins, lipids, and DNA. This con-glomeration of damage can alter the geneticcode, as well as irreversibly injure cells and tissue.Fig. 2.3 UV attenuation vs. wavelength for spherical TiO2of varying particle size. Blue line, 20 nm; green line, 50 nm;and red line,100 nm
132 Nanotechnology in PhotoprotectionThe potential phototoxicity of TiO2nanoparticleswas ﬁrst demonstrated as DNA damage fromhydroxyl radicals (·OH) after exposure to UV irra-diation [7–10]. In the presence of free radicalquenchers such as dimethyl sulfoxide and manni-tol, the DNA damage could be suppressed. Thetarget damage included oxidative biomarkers suchas 8-oxoguanosine , DNA strand breakage [7,10], and structural chromosomal aberrations .TiO2and ZnO in combination had similar geno-toxic effects . Further in vitro studies demon-strated that the DNA damage resulting from TiO2and ZnO agents are cytotoxic in the presence ofcell cultures [11–13]. By using the technique ofelectron spin resonance spin trapping to catchthese short-lived, volatile molecules, other ROShave been proposed to be generated in the process:hydrogen peroxide (H2O2) [14, 15], superoxideanion (O2−) , singlet oxygen (1O2) [16, 17], andcarboxyl radical anion (·CO2−) . The size andcrystal form of the metal oxide also affects toxic-ity: the anatase form of TiO2is more effective atgenerating ·(middot) OH radicals than the rutileform . These studies suggest that metal oxidesare phototoxic by way of generating free radicalspecies upon UV irradiation. In order to determinewhether the phototoxicity was truly due to thepresence of metal oxides alone, Dufour et al. studied mammalian cells to examine the ability ofZnO to damage DNA in the presence of UV lightwhen compared to its activity in the dark. Theauthors measured the effects of ZnO on Chinesehamster ovarian cells under three conditions: inthe dark, pre-irradiation followed by ZnO treat-ment (PI) and simultaneous irradiation with ZnOtreatment (SI). Interestingly, the nature, incidence,and severity of chromosomal aberrations (CA) inthe PI and SI group were nearly identical.Therefore, the timing of exposure of the test agentand cells to UV radiation appeared to have hadno effect on the frequency of CA induction. Thisis in contrast to true photoclastogenic agents, inwhich clastogenic potency would be signiﬁcantlyhigher under simultaneous irradiation conditions.Therefore, the authors concluded that UV radia-tion mediated enhanced susceptibility of mam-malian cells to ZnO, but ZnO itself wasnon-photoclastogenic.There are several arguments that support thesafety proﬁle of nanoparticles. In the previousstudies, the behavior of nanoparticles has onlybeen elucidated in vitro, not in a true biologicalsetting. In fact, the skin has in place an elaborateantioxidant mechanism, composed of enzymaticand nonenzymatic molecules, to quench ROS.Therefore, ROS generated by TIO2and ZnO dur-ing UV exposure can be neutralized by the body’snatural defense mechanism. Besides endogenousprotection, there are numerous synthetic tech-niques which have been developed to reducethese potential risks and decrease the reactivity ofthese nanoparticles. Pﬂucker et al.  ; LivraghiS, et al.  tested a carbon coating on TiO2pow-der and demonstrated signiﬁcant reductions inthe formation of superoxide anion (·O2−) andhydroxyl radicals (·OH), even after UV exposure.Pan et al.  grafted an organic coating whichcould prevent nanoparticle adherence to the cellmembranes of human dermal ﬁbroblasts, andeffectively reduce the cytotoxicity of ROS. Othertechniques such as manganese doping and hydro-phobic polymers have been proposed to preventthe contact of the TiO2surface with oxygen andwater to inhibit the formation of radical species[22, 23]. The ﬁnal argument in support of nano-technology in sunscreens is the overall safetyrecord of TiO2and ZnO. Both metal oxides havebeen used in mineral sunscreens and other topicalproducts for years, with no studies demonstratingadverse effects of potentially harmful free radi-cals. TiO2can be found in toothpaste, lotion,skimmed milk, and cottage cheese, and ZnO is amajor component in many baby powders, anti-dandruff shampoos as well as barrier creams.Another component of the safety of nanopar-ticles is predicated on the dermal penetration ofthese materials and whether these small-sizedminerals can penetrate the human skin. Are therenew risks of cosmetic formulations containingnanosized particles when compared to traditionalingredients? The obvious concern is that thesmaller particle size can enhance skin penetra-tion, pass the skin barrier, and increase the riskfor systemic toxicity and exposure. For mostsubstances, the stratum corneum (SC) is the rate-limiting barrier against the percutaneous penetration
14 L.L. Chen et al.of topically applied substances. This non-viableoutermost layer of the epidermis is approximately15–20 mm thick in humans. Nanoparticles mustpass the SC to reach the living skin in order toexert its toxicities. Most studies done underin vitro or in vivo conditions must be interpretedcautiously for several reasons. Animal skin sam-ples have differing permeability for certain mate-rials. In general, the order from most permeableto least permeable is rabbit, rodent, pig, andlastly, human skin. In addition, penetration ofsubstrates may be favored if skin occlusion meth-ods (which increases swelling of the corneocytes)were used in the studies. Finally, destructionmethods such as tape stripping of the skin mayartiﬁcially capture particles in the deep hair fol-licles or skin furrows to incorrectly conclude thata substance has penetrated the epidermis. In lightof these study limitations, the consistent ﬁndingsfrom these studies suggest that TiO2and ZnOnanoparticles remain on the surface of the SC andare unable to reach the living skin layers(Tables 2.1, 2.2, 2.3). Most studies have reportedthat nanoparticles only penetrate into hair follicleopenings and skin furrows, with negligible mate-rials found below the SC. In 2000, the EUScientiﬁc Committee on Cosmetics and Non-Food Products (SCCNFP) summarized a seriesof ten studies all investigating the percutaneouspenetration of nanosized TiO2pigments .Nanoparticles were detected using a myriad ofimaging techniques including electron and lightmicroscopy, X-ray ﬂuorescence, particle-inducedX-ray emission (PIXE), and electron emissionspectrophotometry. All studies concluded thatnanosized TiO2particles remain on the outer lay-ers of the SC and do not penetrate into the livingskin. Similarly, ZnO nanoparticles showed negli-gible penetration beyond the SC in animal andhuman skin studies. These skin penetration stud-ies consistently summarize that neither nanopar-ticle can penetrate beyond the SC.A number of factors may explain the poorpenetration through the stratum corneum ofintact and healthy human skin. From the ﬁeld oftransdermal drug delivery, it is known that theTable 2.1 TiO2 skin penetration studies (Adapted with permission from Newman et al. )Study Material Particle size Skin model/design ResultsTan et al.  TiO2(no coatingspeciﬁed)Not speciﬁed Human skin,in vitroNo signiﬁcant penetration intoskinLademannet al. TiO2(Al2O3, stearicacid coated)150–170 nm Human skin biopsy Penetration into upper layers ofstratum corneum; ~1 % ofparticles in ostium of follicleEuropeanUnion SCCNFPOpinion TiO2(anatase andrutile forms, variouscoatings)14–200 mm Pig skin, in vitroHuman skin, tapestripping, or biopsyNo penetration beyond thestratum corneum in any studyPﬂuckeret al. TiO2(SiO2, Al2O3,Al2O3+SiO2coated)10–100 nm Human skin biopsy Penetration into upper layers ofthe stratum corneumSchulzet al. TiO2(SiO2±Al2O3coated10–100 nm Human skin biopsy Penetration into the upper layersof the stratum corneumGottbrath andMuller-Goymann TiO2-containingsunscreen (no coatingspeciﬁed)Not speciﬁed Human skin, tapestrippingParticles into upper layers ofstratum corneumMenzelet al. TiO2(various forms,no coating speciﬁed)45–150 nm Pig skin, in vitro Particles in stratum corneum;minimal penetration into stratumgranulosumPopov et al.  TiO2(rutile form) 100 nm Human skin, tapestrippingNo penetration beyond thestratum corneumMavonet al. TiO2(SiO2coated) 20 nm Human skin, tapestripping andin vitroPenetration in upper layers ofstratum corneum
152 Nanotechnology in PhotoprotectionTable 2.2 ZnO skin penetration studies (Adapted with permission from Newman et al. )Pirot et al, 1996 ZnO (no coatingspeciﬁed) Not speciﬁed Human skin, in vitro 0.36 % penetration in 72 hEuropean UnionSCCNFPOpinion ZnO Not speciﬁed Pig skin, in vitro No increase in plasma zinc levels;in vitro, penetration <1 % ofdose; most ZnO recovered fromstratum corneumHuman nonpsoriaticand psoriatic skinCross et al.  ZnO (siliconatecoated)15–30 nm Human skin, in vitro <3 % of applied Zn recovered instratum corneum; penetration intoupper layers of the stratumcorneumZvyagin et al.  ZnO (uncoated) 26–30 nm Human skin, in vivoand in vitroNo penetration beyond stratumcorneum, accumulation in skinfolds and hair folliclesTable 2.3 Combination TiO2 and ZnO skin penetration studies (Adapted with permission from Newman et al. )Lansdown andTaylor TiO2, ZnO(no coatingspeciﬁed) <2–20 mm Rabbit skin, in vivoPenetration into stratumcorneum and outer hairfollicleDussert et al.TiO2, ZnO(no coatingTiO2: 50–100 nm Human skin, in vitro Penetration into upperlayers of stratumZnO: 20–200 nmGontier et al.TiO2, ZnO (Al2O3) Not speciﬁed Human, pig, mouse skin,in vitroTiO2found in intercel-lular space betweencorneocytes of upperlayers of stratumcorneumGamer et al.TiO2(SiO2,dimethiconecoated)TiO2: 30–60 nm Pig skin, in vitro Penetration into upperlayers of stratumcorneum; 0.8–1.4 % ofapplied dose recoveredZnO (uncoated) ZnO: <160 nmFilipe et al.  TiO2(SiO2±Al2O3-coatedTiO2: 20 nm Human skin (intact,compromised, andpsoriatic), tape strippingNo penetration beyondstratum corneumZnO: 20–60 nmZnOupper limit of molecular size of a drug capableof skin penetration is 500 Da (2.5 nm) [10, 25].Common dermatological drugs such as corticos-teroids and antifungals all fall below this sizeand can penetrate the stratum corneum. As men-tioned, nanosized TiO2and ZnO exist as aggre-gates and agglomerates in sunscreen products,their ﬁnal size often exceeding 100 nm, 40 timeslarger than the upper limit. Besides molecularsize, the drug substance must meet the criteriafor polarity, concentration, and melting tempera-ture. Nanoparticles are also considered insolublesubstances, lacking a diffusion driving force topromote penetration into the skin. Besides thephysiochemical properties of nanoparticles, thelocal skin environment may be preventative ofskin penetration. The stratum corneum is con-stantly turning over and this constant sheddingprocess prevents long-term accumulation andpenetration of nanoparticles into the viablecomponents of the skin tissue. Finally, there is apotential penetration pathway of nanomaterialsthrough the follicles, ﬁrst proposed by Lademannet al. . Hair follicles, with its tight network
16 L.L. Chen et al.of capillaries, are an important target for trans-dermal drug delivery and potential nanoparticleuptake. TiO2nanoparticles were found collectingin the “follicular sink” (the hair follicle openingsand superﬁcial portion of the follicles) at con-centrations two orders of magnitude smaller thanthe upper part of the stratum corneum. However,it was later determined that the penetration pro-cess depends on a certain phase of the hairgrowth cycle; nanoparticles are only found whenhair growth and sebum production are active.Therefore, despite the discovery of nanomateri-als in the follicular oriﬁces, the potential forthem entering the living skin tissue is negligible,mainly because growing hair shafts push thesematerials to the surface of the skin [27, 28].It is hypothesized that the percutaneousabsorption of topically applied substances will bealtered if the skin barrier is compromised or dis-rupted. In general, it is unknown what degree ofcompromised skin integrity will signiﬁcantlyincrease skin penetration of small-sized particles.In hyperkeratotic skin conditions such as psoria-sis vulgaris, a thicker epidermis actually enhancesthe skin’s barrier function, which reduces pene-tration of topical drugs. Similarly, it has beenfound that there is identical or lower percutane-ous absorption on intact compared to inﬂamedskin (from UVB-induced sunburn) followingapplication of a [14C]methylprednisolone ace-ponate-containing lotion. The percutaneousabsorption increased after skin tape stripping(removal of the stratum corneum) . Skin con-ditions which disrupt the skin barrier such aseczema have increased penetration of topicallyapplied substances . In a recent study of foursunscreen formulations containing nanosizedTiO2and ZnO (primary particle size 10–50 and140 nm, respectively), UVB irradiation slightlyenhanced the SC penetration of both types ofnanoparticles in pig skin (TiO2deeper than ZnO).However, there was no evidence of systemicabsorption . Though a large amount of stud-ies has rendered nanoparticle safety in cosmeticproducts in normal skin, their safety should befurther assessed taking into account abnormalskin conditions and the possible impact ofmechanical effects on skin penetration.2.6 ConclusionSunscreen will continue to be an important com-ponent of photoprotection. The advancement innanotechnology has allowed nanosized TiO2andZnO ingredients to be amply incorporated intomore effective and cosmetically acceptable sun-screen products. In developing modern inorganicsunscreens based on nanoparticles, formulatorsneed to understand the UV-visible properties ofTiO2and ZnO nanomaterials in relation to theirparticle size. A systemic and methodical approachis required to maintain the optimal size of thesenanoparticles in the ﬁnal sunscreen formulation.The safety debate is ongoing, despite overall con-sistent ﬁndings from laboratory studies demon-strating no penetration of nanoparticles into theliving layers of the skin. Despite their inert behav-ior, nanoparticles could still pose a risk on com-promised, sun damaged skin or from inhalation(such as from spray sunscreens). Such questionsremain to be answered.References1. Mitchnick MA, Fairhurst D, Pinnell SR. Microﬁne zincoxide (Z-cote) as a photostable UVA/UVB sunblockagent. J Am Acad Dermatol. 1999;40(1): 85–90.2. Therapeutic Goods Administration. A Review of thescientiﬁc literature of the safety of nanoparticulatetitaniumdioxideorzincoxideinsunscreens.AustralianGovernment Department of Health and Ageing,2006.3. Popov AP, et al. Effect of size of TiO2nanoparticlesembedded into stratum corneum on ultraviolet-A andultraviolet-B sun-blocking properties of the skin.J Biomed Opt. 2005;10(6):064037.4. Cross SE, et al. Human skin penetration of sunscreennanoparticles: in-vitro assessment of a novel micron-ized zinc oxide formulation. Skin Pharmacol Physiol.2007;20(3):148–54.5. Nohynek GJ, et al. Grey goo on the skin? Nano-technology, cosmetic and sunscreen safety. Crit RevToxicol. 2007;37(3):251–77.6. Newman MD, Stotland M, Ellis JI. The safety ofnanosized particles in titanium dioxide- and zincoxide-based sunscreens. J Am Acad Dermatol. 2009;61(4):685–92.7. Dunford R, et al. Chemical oxidation and DNA dam-age catalysed by inorganic sunscreen ingredients.FEBS Lett. 1997;418(1–2):87–90.
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