European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105

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European Journal of Pharmaceutics and Biopharmaceutics

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Research Paper

Cutaneous penetration of soft nanoparticles via photodamaged skin:Lipid-based and polymer-based nanocarriers for drug delivery

http://dx.doi.org/10.1016/j.ejpb.2015.05.0050939-6411/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Pharmaceutics Laboratory, Graduate Institute ofNatural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan,Taoyuan 333, Taiwan.

E-mail address: fajy@mail.cgu.edu.tw (J.-Y. Fang).1 Equal contribution.

Chi-Feng Hung a,1, Wei-Yu Chen b,c,1, Ching-Yun Hsu d, Ibrahim A. Aljuffali e, Hui-Chi Shih f,g,Jia-You Fang f,h,i,⇑a School of Medicine, Fu Jen Catholic University, Hsinchuang, New Taipei City, Taiwanb Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwanc Department of Pathology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwand Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwane Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabiaf Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwang Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwanh Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwani Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 February 2015Revised 7 May 2015Accepted in revised form 11 May 2015Available online 15 May 2015

Keywords:PhotodamageSkinCutaneous penetrationLipid nanoparticlePolymer nanoparticle

Photoaging is recognized as the factor damaging skin-barrier function. The aim of this study was to exam-ine the impact of ultraviolet (UV) irradiation on the cutaneous penetration of soft nanoparticles, includingnanostructured lipid carriers (NLCs) and poly(lactic-co-glycolic acid) polymer nanoparticles (PNs). Invitro cutaneous permeation of retinoic acid (RA) carried by nanoparticles was evaluated. In vivo nudemouse skin distribution of topically applied nanoparticles was observed by fluorescence and confocalmicroscopies. The association of nanoparticles with cultured keratinocytes was measured by flow cytom-etry and fluorescence microscopy. The average diameter and surface charge were 236 nm and�32 mV forNLCs, and 207 nm and �12 mV for PNs. The ultrastructural images of skin demonstrated that the appli-cation of UV produced a loss of Odland bodies and desmosomes, the organelles regulating skin-barrierfunction. UVA exposure increased skin deposition of RA regardless of nanoparticle formulation. UVBdid not alter RA deposition from nanoparticles as compared to the non-treated group. Exposure toUVA promoted RA delivery into hair follicles from NLCs and PNs by 4.2- and 4.9-fold, respectively. Thein vivo skin distribution also showed a large accumulation of Nile red-loaded nanoparticles in folliclesafter UVA treatment. The soft nanoparticles were observed deep in the dermis. PNs with higherlipophilicity showed a greater association with keratinocytes compared to NLCs. The cell association ofPNs was increased by UVA application, whereas the association between NLCs and keratinocytes wasreduced two times by UVA. It was concluded that both follicles and intercellular spaces were the mainpathways for nanoparticle diffusion into photodamaged skin.

� 2015 Elsevier B.V. All rights reserved.

1. Introduction

Nanomedicine provides potential benefits in the fields of drugdelivery, medical diagnosis, cancer therapy, and tissue engineering[1]. The growth of nanomedicine has led to the translational devel-opment of industrial techniques and consumer products.

Functional nanoparticles have gained much attention in targetedand controlled drug delivery. Within this field, the skin is the pre-dominant target organ for nanoparticle exposure. The drug-loadednanocarriers are an effective approach to target skin regions andcell populations of interest [2]. The impairment of skin integrityis becoming significant due to today’s lifestyle and severe climatechange. The barrier function of skin exposed under occupationalor environmental conditions is not ideal. Ambient particulate mat-ters, air pollution, and solar radiation all pose the risk ofskin-barrier damage [3]. It is anticipated that nanoparticle deliveryvia damaged skin is quite different from that via intact skin. A

C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105 95

comprehensive elucidation of nanoparticle-skin interaction fordamaged skin is highly relevant.

The thinning of the ozone layer has produced increased ultravi-olet (UV) irradiation of the skin and the subsequent syndromes ofphotodamage in the past few decades. The impact of photoaging onnanoparticle penetration into the skin has already been studied byusing quantum dots (QDs), titanium dioxide nanoparticles, andfullerenes [4–7]. These nanoparticles can be categorized as rigidnanoparticles according to the definition by Papakostas et al. [8].Rigid nanoparticles are generally made of inorganic materials withcrystalline and solid properties, while soft nanoparticles are madeof organic materials (e.g. lipids, polymers, and proteins) withdeformable characters when undergoing stress or contacting withsurface. However, there is a lack of study of soft nanoparticle pen-etration of photodamaged skin with respect to lipid-based andpolymer-based systems. The goal of this study was to comparethe permeation of soft nanoparticles via healthy and photodam-aged murine skin. If the nanoparticles can deliver into the skin tis-sues, they interact with the local cellular environment. In thepresent work, we evaluated the interaction between soft nanopar-ticles and skin with regard to skin permeation and keratinocyteuptake.

Nanostructured lipid carriers (NLCs) consisting of liquid andsolid lipids as the inner matrix were employed as a model oflipid nanoparticles in this study. NLCs are extensively used formedical and cosmetic products with good drug targeting,improved drug stability, and easy scale-up [9]. We selectedpoly(lactic-co-glycolic acid) (PLGA) as the material to preparepolymer nanoparticles (PNs) due to its high biocompatibility andthe approval by the USFDA and the European Medicine Agencyfor application in drug delivery systems [10]. The model drug usedwas all-trans retinoic acid (RA, tretinoin) because of its wide use forinclusion in topically applied nanoparticles [11]. Topical RA is thefirst drug therapy approved by the USFDA to treat photoaging[12]. It is also utilized for topical treatment of wrinkling, psoriasis,acne, and skin tumors [13]. The topical RA administration cancause skin irritation and peeling. The photostability of RA is alsopoor. RA incorporation into nanoparticles can resolve these short-comings. We examined the in vitro and in vivo RA absorption inthe skin under the influence of photoaging. The fluorescent dyewas also included in the nanoparticles in order to see the distribu-tion within the skin. Finally, the association of nanoparticles withthe cultured keratinocytes was evaluated in the presence andabsence of UV irradiation.

2. Materials and methods

2.1. Materials

RA, squalene, Pluronic F68, PLGA (50:50, molecular weight 30–60 kDa), poly(vinyl alcohol) (PVA), Nile red, and rhodamine 800were purchased from Sigma–Aldrich (St. Louis, MO, USA).Precirol� ATO 5 was provided by Gattefossé (Gennevilliers,France). Soy phosphatidylcholine (SPC, Phospholipon� 80H) wasobtained from American Lecithin (Oxford, CT, USA).

2.2. Fabrication of NLCs

Squalene (900 mg), Precirol� (300 mg), and SPC (20 mg) weremixed as the lipid phase, while the aqueous phase consisted ofPluronic F68 (240 mg) and double-distilled water. RA was addedin the lipid phase as a 40 mg dose. The total volume of the nanodis-persion was 10 ml. Both phases were heated separately to 85 �C for15 min. The aqueous phase was added to the lipid phase, and thenmixed with a high-shear homogenizer (Pro 250, Pro Scientific,

Monroe, CT, USA) at 12,000 rpm for 10 min. A probe-type sonicator(VCX 600, Sonics and Materials, Newtown, CT, USA) was used tofurther treat the nanodispersion for 10 min to fabricate the finalproduct.

2.3. Fabrication of PNs

PLGA (100 mg) and RA (40 mg) were dissolved in acetone (3 ml)as the organic phase. The aqueous phase consisted of PVA (240 mg)in double-distilled water (12 ml). The aqueous phase was heated at85 �C for 15 min, then mixed by the probe-type sonicator for 5 min.After a cooling time of 15 min, the organic phase was added bydrops to the aqueous phase. Subsequently, the mixture was mixedwith a magnetic stirrer at 300 rpm overnight. After the evaporationof the organic solvent, the mixture was centrifuged in an Amicon�

Ultra-15 tube at 900 rpm for 30 min. After the removal of thesupernatant, the nanodispersion was reconstituted usingdouble-distilled water up to 10 ml.

2.4. Size and zeta potential

The mean diameter (z-average) and zeta potential of thenanoparticles were measured by a laser-scattering technique(Nano ZS90, Malvern, Worcestershire, UK). The nanodispersionwas diluted 100-fold with water before the measurement.

2.5. Encapsulation efficiency of RA

The percentage of RA loading in the nanoparticle matrix wasmeasured using an ultracentrifugation method. The nanodisper-sion was centrifuged at 48,000g at 4 �C for 30 min. The supernatantand precipitate were separated and analyzed by high-performanceliquid chromatography (HPLC) described previously [14].

2.6. Molecular environment

The solvatochromism of Nile red in nanoparticles can act as anindicator for demonstrating the polarity of the nanodispersion. Thenanosystem with Nile red (1 ppm) was prepared as described ear-lier. The emission fluorescence spectrum was examined with a flu-orescence spectrometer. The kex and kem were set at 546 and 550–700 nm, respectively. The scanning speed was 300 nm/min.

2.7. Animals

Eight-week-old female nude mice (JCR-Foxn1nu) were suppliedby the National Laboratory Animal Center (Taipei, Taiwan). Theanimal experimental protocol was reviewed and approved by theInstitutional Animal Care and Use Committee of Chang GungUniversity. Ethical issues with animal experiments complied withDirective 86/109/EEC from the European Commission.

2.8. UV exposure

A Bio-Sun Illuminator (Vilber Lourmat, Marne-la-Vallée, France)was employed to produce the radiation of UVA (365 nm) or UVB(312 nm). The distance between the dorsal skin of nude mice andthe UV lamp was 10 cm. The spectral irradiance was 9 J/cm2 and190 mJ/cm2 for UVA and UVB, respectively. The dorsal area wasexposed with UVA every other day for five days. UVB treatmentwas performed once a day for five days.

2.9. Physiological examination

Transepidermal water loss (TEWL) and skin lightness (L⁄) of thedorsal skin were measured 1 h after the completion of the UV

96 C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105

irradiation course. A Tewameter� (TM 300, Courage and Khazaka,Köln, Germany) was employed for TEWL detection. A spectrocol-orimeter (CD 100, Yokogawa, Tokyo, Japan) was used to quantify L⁄.

2.10. Ultrastructural examination by transmission electron microscopy(TEM)

The dorsal skin excised from nude mice was cut intoappropriate-sized cubes and then fixed at 4 �C in 2% paraformalde-hyde and 2.5% glutaldehyde in 0.2 M cacodylate and 7% sucrosebuffer for 15 min, postfixed with 2% osmium tetroxide for 24 h.The other procedure was the same as described previously [15].The skin slice was observed using Hitachi H-6000 TEM (Tokyo,Japan).

2.11. In vitro cutaneous penetration

This experiment was conducted with Franz diffusion cells. Theexcised skin without any treatment or with UV treatment wasmounted between the donor and receptor compartments withthe stratum corneum (SC) facing upward into the donor. The recep-tor contained 30% ethanol in pH 7.4 buffer with a volume of 5.5 ml.The donor (0.5 ml) was filled with RA-loaded nanoparticles or thecontrol solution. The medium for dissolving RA in the control solu-tion was 40% propylene glycol (PG) in pH 7.4 buffer. The effectivediffusion region for penetration was 0.785 cm2. The temperatureand stirring rate of the magnetic stirrer were kept at 37 �C and600 rpm, respectively. At determined intervals, a 300-ll mediumwas withdrawn from the receptor. After a 24-h application, theskin was removed from the Franz cell. An RA amount within thecutaneous reservoir was extracted by methanol.

RA uptake by hair follicles was examined by using a cyanoacry-late skin surface casting technique. Subsequent to stripping the SCby adhesive tape, a follicular cast was prepared. A drop of super-glue was added on a glass slide, which was pressed onto the sur-face of the SC-stripped skin. The cyanoacrylate polymerized, andthe slide was expelled with one quick movement after 5 min. Thesuperglue remaining on the slide was scraped off and positionedin a test tube with 2 ml methanol. The tube was shaken for 3 h.The final product was vacuumed to evaporate the methanol. Themobile phase was incorporated to dissolve the residual. All sam-ples obtained from the in vitro penetration experiment were ana-lyzed by HPLC.

2.12. In vivo cutaneous penetration

A glass cylinder with a hollow area of 0.785 cm2 was attached tothe dorsal region of the mouse by superglue. An aliquot of 0.2 ml ofnanoparticles or control solution containing Nile red (0.01% w/v)was pipetted into the cylinder. The application period was 6 h.The animal was then sacrificed, and the treated skin area wasexcised. Skin biopsies were sliced by a cryostat microtome, embed-ded in OCT, and frozen at �80 �C. Subsequently the samples weremounted with glycerol and gelation. The specimens were moni-tored by fluorescence microscopy (IX 81, Olympus, Tokyo, Japan)using a filter of 450–490 nm for kex and 515–565 nm for kem. A con-focal laser scanning microscope (TCS SP2, Leica, Wetzlar, Germany)was used to observe a horizontal section of the skin. The thicknessof the skin was scanned at about 5 lm increments via z-axis.Images were taken by summing 15 fragments at different depthsfrom the skin surface.

2.13. Cell viability assay

The spontaneous immortalized human keratinocytes (HaCaTcell line) were a gift from Dr. Y. J. Lee, Fu Jen Catholic University.

The cell culture process was described in our previous report[16]. The keratinocytes (3 � 104 cells/well) were seeded in a24-well plate. The nanoparticles with different dilutions wereincubated with keratinocytes for 24 h. After a brief wash withmedium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) at a concentration of 0.5 mg/ml in DMEM was usedfor the measurement of living metabolically active cells.Mitochondrial dehydrogenases metabolized MTT to a purple for-mazan dye, which was determined by a UV spectrophotometer at550 nm.

2.14. Flow cytometry

The keratinocytes (3 � 104 cells/well) in the plate were exposedto UVA or UVB by a Bio-Sun illuminator at an irradiation dose of15 J/cm2 or 10 mJ/cm2, respectively. Immediately after UV treat-ment, the nanoparticles at a concentration of 20 ll/ml were incu-bated with HaCaT for 2 h. Rhodamine 800 (0.01% w/v) in thenanodispersion was employed as fluorescent dye for flow cytome-try detection. After a brief wash, the cells were collected by scrap-ing and centrifugation. The cell pellets were reconstituted by PBS(1 ml) and then analyzed by a flow cytometer (CyFlow ML,Partech, Münster, Germany) at kex and kem of 488 and 525 nm,respectively.

2.15. Nanoparticle-keratinocyte association observed by fluorescencemicroscopy

The cell uptake of the nanoparticles by HaCaT was examinedafter treatment with UV irradiation. The uptake was terminatedby cell washing with ice-cold PBS three times. 40,6-Diamidino-2-phenylindole (DAPI) was used to stain the cell nucleus. The proce-dure was performed based on the previous study [17]. The imageswere obtained using fluorescence microscopy (IX 81, Olympus).

2.16. Statistical analysis

Statistical analysis of the differences between the groups wasperformed using the Kruskal–Wallis test. The post hoc test usedfor checking individual differences was Dunn’s test. A 0.05 levelof probability (p < 0.05) was used as the level of significance.

3. Results

3.1. Physicochemical characterization of the nanoparticles

As summarized in Table 1, NLCs had a diameter of 236 nm and apolydispersity index (PDI) of 0.37 with a surface charge of �32 mV.The PNs presented a size of 207 nm and the value of PDI was 0.11.The preparation of PNs contributed to a negative surface charge of�12 mV. RA incorporation into nanoparticles at an encapsulationpercentage of 82% was achieved by NLCs. A greater RA loadingcapacity was obtained by PNs, showing a percentage of 98%. Thepolarity (molecular environment) of NLCs and PNs was comparedby examining Nile red emission as illustrated in Fig. 1. Nile redcan be largely dissolved in a lipophilic environment with anintense fluorescence emission. PNs revealed stronger fluorescenceintensity than did NLCs in the wavelength range between 550 and700 nm. This indicates a greater lipophilicity of PNs than NLCs.

3.2. Physiological and ultrastructural examinations

The physiological changes by UVA and UVB were determinedbased on TEWL and skin lightness. As depicted in Fig. 2, UVAinduced a significantly higher level (p < 0.05) of TEWL

Table 1The characterization of NLCs and PNs by particulate size (nm), polydispersity index(PDI), zeta potential (mV), and RA encapsulation percentage (%).

Formulation Size (nm) PDIa Zetapotential(mV)

RA encapsulationpercentage (%)

NLCs 236.0 ± 4.1 0.37 ± 0.04 �31.6 ± 2.0 81.5 ± 1.5PNs 207.2 ± 3.0 0.11 ± 0.04 �12.2 ± 1.7 98.0 ± 0.6

All data are presented as the mean of three experiments ± S.D.a PDI, polydispersity index.

NLCs PNs

Wavelength (nm)

Inte

nsity

(A.U

.)

Fig. 1. Fluorescence emission spectra of Nile red (1 ppm) in nanostructured lipidcarriers (NLCs) and polymer nanoparticles (PNs). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web versionof this article.)

C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105 97

(17.17 g/cm2/h) than in the non-treated control (7.41 g/cm2/h).UVB further augmented TEWL to 24.04 g/cm2/h. However, thisvalue did not reach a statistical significance (p > 0.05) comparedto the value of the UVA group. The UVA-treated area developed awhitened skin color based on the L⁄ measurement shown inFig. 2B. UVB did not alter (p > 0.05) the lightness of the mouse skincompared to the control. The ultrastructural analysis of the SC andstratum granulosum (SG) by TEM in intact and photodamaged skinwas carried out. We examined the presence of Odland bodies anddesmosomes since they are essential to constructing the barrierfunction. There were some Odland bodies with an ovoid shapeencircled in the SC of intact skin as shown in the left panel ofFig. 2C. The right panel of Fig. 2C shows the appearance of SG lay-ers. The desmosomes could be clearly observed in this region.When considering the skin treated by UVA (Fig. 2D), a distinct dif-ference in skin structure was apparent. The number of Odland bod-ies markedly decreased after UVA irradiation. The number ofdesmosomes in the SG also decreased following UVA exposure.We observed expansion of the interstices in the cell junctions,which could be recognized as intercellular edema. As shown inFig. 2E, no Odland bodies or desmosomes were found in the skinof the UVB group.

3.3. In vitro cutaneous penetration

The in vitro permeation of RA into/across nude mouse skin wasdetermined in terms of skin deposition, flux, and follicular amount.Table 2 shows the data of free RA in 40% PG aqueous solution. Thecutaneous deposition of RA into intact skin was 15 lg/mg. UVArevealed an increased RA deposition of 2.7-fold over intact skin.The RA accumulation for UVB-treated skin was not changed to

any appreciable degree (p > 0.05). RA penetration into the receptorfollowed zero-order kinetics. The same as skin accumulation,UVA-treated skin was much more permeable to RA than intactskin. A 2.8-fold increase in RA flux was achieved after UVA expo-sure. On the other hand, UVB exposure significantly decreasedthe RA flux from the control solution (p < 0.05). UVA showed a1.8-fold greater RA uptake in follicles compared to that ofnon-treated skin. UVB produced a significant reduction in follicularRA (p < 0.05) from 2.01 to 1.23 lg/cm2.

Table 3 presents the cutaneous penetration profiles of RA fromNLCs. The RA deposition in intact skin from NLCs was 28.3 lg/mg,which was higher (p < 0.05) than that from the control solution.Application of UVA and UVB increased the cutaneous reservoir2.1- and 1.2-fold over the uptake from non-treated skin.However, there was no significant difference (p > 0.05) betweenthe skin deposition of the nontreatment and UVB groups. The fluxfrom NLCs was in the order of UVA > UVB P nontreatment, thesame as detected for cutaneous deposition. UVA triggered greaterRA uptake into the hair follicles by a factor of 4.2. The extent of fol-licular RA uptake by UVB treatment was lower (p < 0.05) comparedto non-treated skin (0.09 versus 0.15 lg/cm2).

As shown in Table 4, polymer nanocarriers had a 2.4-fold higheraccumulation in intact skin compared to that of aqueous solution.RA deposition from PNs provided by UVA application was higher(p < 0.05) than that by nontreatment and UVB. RA flux acrossUVA- and UVB-treated skin from PNs was not significantly morethan that across healthy skin (p > 0.05). The follicular RA uptakefrom PNs displayed a similar tendency with skin accumulation,with the UVA group showing the highest uptake followed by theUVB and nontreatment groups.

3.4. In vivo cutaneous penetration

The in vivo skin absorption of nanoparticles was tracked by flu-orescence and confocal microscopies. Fig. 3 shows the nanoparticledistribution in the vertical cross sections of skin with or withoutUV application. The images under visible light, fluorescence light,and the merging are illustrated in this figure. As depicted inFig. 3A, a negligible fluorescence was observed in the intact skinafter topical administration of NLCs. A stronger level of fluores-cence was distributed to viable skin after UVA irradiation. Theimage apparently indicated a preferential accumulation of lipidnanoparticles in the follicles. UVB slightly increased the red signalin the skin treated by NLCs. The red staining was evident in theareas of the appendages. In the intact skin, PNs-associated fluores-cence was mainly observed in the epidermis and hair follicles asshown in Fig. 3B. The fluorescence intensity of healthy skin admin-istered by PNs was stronger than that by NLCs. UVA facilitated thepenetration of PNs into the skin. The fluorescence from PNsshowed a uniform distribution throughout the skin. UVB did notincrease the red signal in the skin compared to the non-treatedgroup. The results from fluorescence microscopy were in agree-ment with the in vitro permeation profiles.

The nanoparticle distribution in the skin by a horizontal viewwas checked using confocal microscopy. We acquired the x–yplaned sectional imaging from the skin surface with a �5-lmincrement. Fig. 4 shows the fluorescence in different layers andthe summary of 15 fragments. The same as fluorescence micro-scopy, UVA and UVB increased the deposition of NLCs as shownin Fig. 4A. UVA augmented red fluorescence mainly in the deeperskin strata but not in the superficial layers. This phenomenonwas also observed in the application of PNs on UVA-treated skin(Fig. 4B). The qualitative analysis of nanoparticle biodistributionrevealed the greater fluorescence intensity from Nile red-loadedPNs after UVB irradiation. This intensity did not surpass that ofUVA-treated skin.

Ligh

tnes

s (L

*)

30

35

40

45

50

55

60

65

Non-treatment UVA UVB

*

TEW

L (g

/m2 /h

)

0

5

10

15

20

25

30

35

UVA UVB

*

*

Desmosome

Stratum granulosumStratum corneum

Odland body

Stratum granulosum

Desmosome

Odland body

Intercellular edema

Odland body (rare)

Stratum corneum

Stratum granulosum

Desmosome (not found)

Stratum corneum

Odland body (not found)

(B)(A)

(D)

(C)

(E)

Fig. 2. The physiological and ultrastructural examinations of nude mouse skin after treatment of UVA and UVB: (A) transepidermal water loss (TEWL), (B) skin lightness (L⁄),(C) transmission electron microscopy (TEM) of stratum corneum (SC) and stratum granulosum (SG) without any treatment (control group), (D) TEM of SC and SG with UVAtreatment, and (E) TEM of SC and SG with UVB treatment.

98 C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105

Table 2Skin accumulation (lg/mg), flux (lg/cm2/h), and follicular amount (lg/cm2) of RApermeation from 40% PG/pH 7.4 buffer via skin treated with UVA and UVB.

Radiation Skin accumulation(lg/mg)

Flux (lg/cm2/h) Follicular amount(lg/cm2)

None 15.01 ± 1.65 0.37 ± 0.09 2.01 ± 0.20UVA 40.25 ± 13.18 (2.68) 1.04 ± 0.22 (2.81) 3.51 ± 1.15 (1.75)UVB 19.55 ± 6.26 (1.30) 0.21 ± 0.05 (0.57) 1.23 ± 0.29 (0.50)

The data in parenthesis behind the mean ± S.D. are the ratios between UV-treat-ment data and non-treatment data.All data are presented as the mean of four experiments ± S.D.

Table 3Skin accumulation (lg/mg), flux (lg/cm2/h), and follicular amount (lg/cm2) of RApermeation from NLCs via skin treated with UVA and UVB.

Radiation Skin accumulation(lg/mg)

Flux (lg/cm2/h) Follicular amount(lg/cm2)

None 28.3 ± 5.55 0.17 ± 0.01 0.15 ± 0.04UVA 59.62 ± 5.19 (2.11) 0.27 ± 0.08 (1.59) 0.63 ± 0.12 (4.20)UVB 34.24 ± 6.02 (1.21) 0.18 ± 0.05 (1.10) 0.09 ± 0.01 (0.60)

The data in parenthesis behind the mean ± S.D. are the ratios between UV-treat-ment data and non-treatment data.All data are presented as the mean of four experiments ± S.D.

Table 4Skin accumulation (lg/mg), flux (lg/cm2/h), and follicular amount (lg/cm2) of RApermeation from PNs via skin treated with UVA and UVB.

Radiation Skin accumulation(lg/mg)

Flux (lg/cm2/h) Follicular amount(lg/cm2)

None 35.63 ± 7.64 0.15 ± 0.01 0.20 ± 0.04UVA 110.22 ± 43.35 (3.09) 0.20 ± 0.03 (1.33) 0.97 ± 0.24 (4.85)UVB 46.23 ± 19.83 (1.30) 0.18 ± 0.04 (1.20) 0.25 ± 0.08 (1.25)

The data in parenthesis behind the mean ± S.D. are the ratios between UV-treat-ment data and non-treatment data.All data are presented as the mean of four experiments ± S.D.

C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105 99

3.5. Nanoparticle-keratinocyte association

We next explored the cellular association of soft nanoparticles.Keratinocytes were selected in this experiment since they are themajor target cells for the photoaging response. At first, the cellsunderwent UVA and UVB irradiation for the measurement of cyto-toxicity. The cell viability was not reduced by UVA and UVB (datanot shown). Fig. 5A presents the cytotoxicity of NLCs and PNs onkeratinocytes with no UV irradiation. Cell viability by the treat-ment of NLCs decreased following the increase of nanoparticle con-centration. A statistical analysis suggested that the doses of 10 and20 ll/ml did not decrease the viability (p > 0.05). NLCs at 80 ll/mlshowed cell viability of 38%. PNs were less cytotoxic than NLCs. Thekeratinocytes remained totally viable after exposure to PNs at allconcentrations tested. A 20 ll/ml dose was chosen for the subse-quent cell association studies to minimize the toxic effect ofnanoparticles.

Flow cytometry allows the quantification of cells associatedwith nanoparticles, including cell uptake and membrane adsorp-tion. Rhodamine 800 was utilized as a dye loading in nanoparticles.UV application did not alter the cell association (p > 0.05) by freerhodamine 800 in DMSO (Fig. 5B). The level of cell association byNLCs in the condition without UV was similar (p > 0.05) to thatby free rhodamine 800. Irradiation of UVA significantly decreased(p < 0.05) keratinocyte association by NLCs by 2-fold. Contrary tothis result, UVB had doubled (p < 0.05) the interaction betweenNLCs and keratinocytes. A dramatic increase in fluorescence inten-sity was detected by PNs, suggesting a strong interaction between

PNs and keratinocytes. The association was significantly greater(p < 0.05) for UVA application in comparison with nontreatment.There was no significant effect (p > 0.05) of UVB on cell interactionwith PNs. The qualitative observation of nanoparticle-cell associa-tion by fluorescence microscopy was performed. No autofluores-cence was detected in the keratinocytes without any treatment(blank), indicating that any fluorescence recorded in the experi-ment originated from the ingestion or adsorption of nanoparticles.Fig. 5C shows the profiles of free rhodamine 800. The red fluores-cence from the groups of non-UV and UV treatments was compa-rable, confirming the results of flow cytometry. We found thatNLCs had a much greater fluorescence signal under UVB irradiationcompared with the nontreatment and UVA groups (Fig. 5D). Thereappeared to be a higher amount of keratinocyte association withPNs than free dye and NLCs (Fig. 5E). Most of the cells were stainedby red fluorescence, especially the cells with UVA exposure. Thisvisualization confirmed the results recognized by flow cytometry.

4. Discussion

It is of interest to know the impact of UV radiation on nanopar-ticle penetration into the skin. Due to climate change in moderntimes, there have been numerous investigations and health warn-ings associated with UV exposure. We aimed to address whetherand to what extent UVA or UVB influenced skin permeation of softnanoparticles, which are widely used in the fields of medical andcosmetic products. Nude mouse skin was the permeation barrieremployed in this work because of its feasibility for studying pho-toaged skin [18,19]. It is also an ideal model for examining follicu-lar accumulation since the follicles of nude/hairless mice areusually undeveloped and degenerated [20]. Their follicles are cov-ered by dry sebum, cell debris, and degenerated corneocytes.About 30–50% of hair follicles in human skin are closed to penetra-tion [21].

The particle diameter of both nanocarriers did not show a largedifference. The influence of the size on the comparison of cuta-neous penetration between NLCs and PNs could be ignored. Thenegative zeta potential of NLCs was higher than that of PNs dueto the negatively charged SPC on the particle shell. The high com-patibility between RA and the nanoparticles contributed to a highentrapment efficiency. PNs showed a greater lipophilicity as com-pared to NLCs according to Nile red emission profiles. This led to asignificantly higher encapsulation of RA in PNs than NLCs since RAis an extremely lipophilic compound with a log P of 6.3. The suffi-cient RA loading in nanoparticles is favorable to minimizing thedirect contact of RA to the skin surface, thus reducing the possibleskin irritation elicited by RA [22].

The cutaneous barrier function can be measured physiologicallyby TEWL. SC and epidermal damage increased TEWL [23]. Ourresults demonstrated that both UVA and UVB promoted TEWL.This phenomenon was especially critical for UVB-treated skinalthough there was no significant difference between the TEWLvalue of the UVA and UVB groups. The UVB-induced TEWL increasewas proved previously [24]. Tsukahara et al. [25] suggested thatUVA can raise TEWL under the conditions of strong energy andprolonged duration. We also examined TEM images ofUV-treated skin to observe Odland bodies and desmosomes, whichplay an important role in barrier-function formation. Odland bod-ies, originating in the SG, are organelles regulating the lipid forma-tion of the skin barrier and maintaining SC stability [26].Desmosomes establish keratinocyte cohesion and intercellularjunctions in epidermal layers [27]. The results showed that UVexposure dramatically reduced the number of Odland bodies anddesmosomes in the SC and SG. The ultrastructural image of skintreated by UVA also displayed an intercellular edema in the SG.

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Fig. 3. The skin distribution with a vertical view of nanoparticles loaded with Nile red after treatment of UVA and UVB: (A) the skin distribution of nanostructured lipidcarriers (NLCs) examined by white light, fluorescence light, and the merging and (B) the skin distribution of polymer nanoparticles (PNs) examined by white light,fluorescence light, and the merging. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

100 C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105

(A)

(B)

Non -treatment UVA UVB

Summary SummarySummary

Non-treatment UVA UVB

Summary SummarySummary

Fig. 4. The skin distribution with a horizontal view of nanoparticles loaded with Nile red after treatment of UVA and UVB: (A) the skin distribution of nanostructured lipidcarriers (NLCs) at different depths and the summary of 15 fragments and (B) the skin distribution of polymer nanoparticles (PNs) at different depths and the summary of 15fragments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105 101

UVA increased skin lightness, which could have been due to theloss of microcirculation supply and necrosis/apoptosis [28].

Damaged skin is expected to exert an increase in cutaneous per-meability compared to healthy skin. Our data confirmed that thenanoparticle skin penetration was enhanced by photodamage,especially for UVA. It is believed that RA and Nile red, the perme-ants used in this study, were transported into the skin in an entrap-ment form within the nanoparticle matrix according to previousstudies [28–30]. RA deposition in the skin could be enhanced byincorporation into NLCs and PNs. The highly specific surface areaof nanoparticles contacting the skin surface could be the main rea-son for this. The assembly of nanoparticles on the skin surfaceforms a thin film and effectively occludes the administration area,resulting in increased SC hydration and the subsequent facility ofdrug diffusion [31]. In the case of NLCs, SPC in the nanoparticleshell can associate with SC lipids, leading to the facile penetrationinto the skin [32]. With respect to PNs, this nanocarrier has a highaffinity to the SC based on the great lipophilicity. The rigidnanoparticles of <20 nm can permeate through the skin in an intactform. This effect is not plausible for NLCs and PNs with a diameter

of >200 nm. The soft nanoparticles may exhibit a flexible confor-mation that can squeeze into the skin. The increase of RA deposi-tion/flux ratio by nanoparticles suggests a preferableaccumulation of nanoparticles within the skin reservoir rather thanpenetration into the aqueous receptor. This effect is beneficial forRA to exert pharmacological activity in the skin and to minimizepossible toxicity in systemic circulation.

Both the SC and epidermis contribute to the permeation resis-tances for nanoparticle delivery. It is assumed that nanoparticlespenetrate into the SC via the intercellular pathway [33]. TheTEWL elevation and the loss of Odland bodies in the SC by photoag-ing indicated the loosening of intercellular space, resulting in theenhanced penetration of nanoparticles. The viable and hydrophilicepidermis also supplies an important barrier for lipophilicnanoparticles. The intercellular space of the epidermis could beexpanded by photoaging due to the loss of desmosomes, a funda-mental regulator of epidermal junctions. Although both UVA andUVB damaged skin integrity, the nanoparticle diffusion was notsignificantly increased by UVB. Our previous studies [28,34] indi-cated that UVB revealed more minor disruption of epidermal

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Fig. 5. The keratinocyte viability and nanoparticle-keratinocyte association after treatment of UVA and UVB: (A) cell viability after treatment of nanostructured lipid carriers(NLCs) and polymer nanoparticles (PNs) at different concentrations, (B) flow cytometry of cell association with NLCs and PNs loaded with rhodamine 800 (R800), (C)fluorescence imaging of cell association with free R800, (D) fluorescence imaging of cell association with NLCs, and (E) fluorescence imaging of cell association with PNs.

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junctions than UVA, including E-cadherin and claudin-1. UVB irra-diation caused increased SC thickness and hyperkeratosis [28,35].The thickened SC and epidermis lengthened the path for nanopar-ticle penetration. The less functional barrier but extended pathwayby UVB caused an insignificant increase in nanoparticlepermeation.

Another observation was the greater RA uptake in follicles afteradministration of the control solution compared to nanoparticles.This could be due to the inclusion of 40% PG in the control solutionwith the aim of dissolving RA. Grice et al. [36] suggested that etha-nol and PG potentially extract the sebum in follicles, thus improv-ing drug delivery into this appendageal route. Nevertheless, it mustbe recognized that the hair follicles are still important for

nanoparticle penetration into the skin because of the natures ofweak barrier function and large reservoirs [37]. The nanoparticleswith a diameter of <300 nm are capable of being internalized bythe follicular canal [38]. Our nanoparticles fitted this criterion.UVA irradiation substantially increased soft nanoparticle uptakeinto the follicles by at least 4-fold. This effect was not detectedfor the UVB group. UVB exposure is predominantly confined tothe superficial region of hair follicles, whereas UVA can penetratedeeply into the follicular cuticles [39]. According to our previousreport [34], the cell debris and desquamated corneocytes inducedby UVB obstructed permeant entry into the follicles. The trend ofnanoparticle uptake by follicles in different skin types was in linewith the cutaneous RA deposition. Hair follicles provided a storage

C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105 103

reservoir for nanoparticles, offering a shortcut for drug release tothe deeper skin strata. PNs exerted a higher follicular RA accumu-lation and skin deposition than NLCs did. The lipophilicity of thedrug carriers is an important factor in managing follicular delivery[40]. The high lipophilicity of PNs raised the interaction withsebum, increasing the depot formation in the follicles.

The cutaneous absorption of rigid nanoparticles such as QDs,titanium dioxide nanoparticles, and zinc oxide nanoparticles intophotodamaged skin was described previously. QDs are metallicnanocrystals composed of cadmium and selenium. Mortensenet al. [4,6] reported that QDs with a diameter of �20 nm accumu-late in the folds of the SC and hair follicles of photodamaged skin. Alow level of QDs can be found in the mouse dermis. The intact QDstend to assemble along the intercellular space among the corneo-cytes. Monteiro-Riviere et al. [5] demonstrated that titanium diox-ide nanoparticles (�200 nm) deeply diffuse into the SC layers ofUVB-damaged pig skin relative to intact skin but do not reachthe epidermal layers. Zinc oxide nanoparticles (�140 nm) remainon the skin surface without any penetration even after UVB irradi-ation. Particle interaction or association with specific structures/-components of the skin is an essential parameter affectingnanoparticle diffusion within the skin. Metallic and lipid/polymernanoparticles show quite different interaction to skin structures[41]. The rigid metallic nanoparticles reach the viable epidermisthrough the lipid matrix of the SC in a complete form. However,this case can be observed only for rigid nanoparticles with a sizeof <36 nm due to the limited dimension of the aqueous pores inthe intercellular route [42]. This is the reason why titanium oxideand zinc oxide nanoparticles stay within the SC layers of photo-damaged skin.

Different from the rigid nanoparticles, no intact form of softnanoparticles penetrates into the skin [33,43]. After penetrationinto the SC with a malleable shape, the soft nanoparticles fuse withthe lipid bilayers. Another difference between metallic andlipidic/polymeric nanoparticles is that the soft nanoparticles canbe biodegradable in the skin [44,45]. It is anticipated that the softnanoparticles exhibit an amorphous form, joining SC componentsafter penetration into the skin (Fig. 6). Honeywell-Nguyen et al.[46] demonstrated that the elastic nanoparticles can reach theSC-viable epidermis junction within 1 h, whereas the rigidnanoparticles are only found in the superficial SC. Although the softnanoparticles could permeate into the SC bilayers, this pathwaywas still a significant barrier even in photodamaged skin. Hair fol-licles are the easy pathway for nanoparticle permeation into theskin. Evidence is that UVA increased both follicular uptake and skindeposition of RA from soft nanoparticles. It is possible that the

Epidermis

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Flexible form into SCExtensive accumulation in follicles

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Fusion with lipids

Release to deeper skin strata

Soft nanoparticlesDrugs

Fig. 6. The possible mechanisms of soft nanoparticle penetration into photodam-aged skin.

fluorescence distribution in the deeper skin strata was derivedfrom the nanoparticles in the hair follicles.

The nanoparticles can easily come into contact with ker-atinocytes and fibroblasts, especially when the skin barrier is dam-aged [47]. Keratinocytes are the most represented cells in theepidermis (90%), which is the first contact layer of the nanoparti-cles when passing into the skin. Previous study [4] indicates thatQDs were taken up by various cell types in the skin after penetra-tion into photodamaged skin. The last section of this study dis-cusses the possible association of nanoparticles withkeratinocytes. The UVA and UVB doses used for this experimentdid not affect the viability of keratinocytes. However, the lipid per-oxidation and injury of the cell membrane may occur in ker-atinocytes exposed to UV [48,49]. We expect damage to thekeratinocyte membrane after UV exposure, although it is beyondthe scope of this study to examine the responses in detail. HaCaTcells favorably ingest nanoparticles or adsorb them on the plasmamembrane in the diameter of <200 nm [47]. Our nanoparticlesshowed a size close to this limitation. The experimental resultsrevealed a higher cell association of PNs than NLCs. The highlipophilicity of PNs supported the interaction of nanoparticles withthe plasma membrane. It is generally recognized that negativelycharged nanoparticles have a low rate of plasma protein adsorp-tion and nonspecific cell internalization [50,51]. The higher nega-tive charge of NLCs compared to PNs contributed to lessassociation of NLCs with keratinocytes. Pinocytosis andclathrin-mediated endocytosis are the possible mechanisms forPLGA nanoparticle internalization into cells [10,52]. UVA, but notUVB, increased the association of PNs with keratinocytes. Thismay indicate that UVA irradiation opened up the endocytosis path-way for PNs. UVA-induced membrane peroxidation might be pref-erential for PNs adsorption.

The existence of SPC on the nanoparticle surface can interactwith or be fused to HaCaT membrane [53]. This is the possiblemechanism for the interaction between NLCs and keratinocytes.The effect of UV on the lipid nanoparticle association with ker-atinocytes was quite different from polymer nanoparticles. UVAand UVB significantly reduced and enhanced the association ofNLCs with cells, respectively. Mortensen et al. [54] indicated thatUVB-treated keratinocytes are more susceptible to the ingestionof QDs. A similar phenomenon existed in the case of NLCs. UVAcan damage the keratinocyte membrane by hydrolysis of arachi-donic acid from membrane phospholipids [55]. The disruption ofa membrane-associated lipid component might lead to the reducedinteraction between SPC and the membrane, resulting in less asso-ciation by UVA. Further investigation is necessary to prove thishypothesis. It can be summarized that NLCs and PNs exhibitedquite different behaviors of keratinocyte association under theinfluence of UV.

5. Conclusions

We reported for the first time, as far as we know, the impact ofUV irradiation on the cutaneous delivery and keratinocyte associa-tion of soft nanoparticles. Both NLCs and PNs had the potential toimprove skin absorption of RA. The drug was mainly confined tothe skin with a reduced penetration through the skin, reflectinglocal targeting to the skin and less systemic exposure from thenanoparticles. UV exposure induced a barrier defect that couldincrease cutaneous deposition of nanoparticles. This effect wasinsignificant for UVB treatment because of the lengthened deliverypathway produced by the thickened epidermis. The follicular routewas shown to be of special relevance for nanoparticle permeationvia UVA-treated skin. Exposure of keratinocytes to soft nanoparti-cles demonstrated the cell uptake or adsorption to the membrane

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with no cytotoxicity. The mechanisms of nanoparticle penetrationinto photodamaged skin are quite different in rigid and softnanoparticles. The rigid nanoparticles can be taken up by the inter-cellular space of damaged skin in an intact form, but the penetra-tion depth is usually restricted to the epidermis. On the other hand,soft nanoparticles exhibited a flexible capability to intrude into andfuse with the lipid matrix of the SC in photodamaged skin. The softnanoparticles could be observed in the dermis of UV-irradiatedskin. Mechanistic insight into how UV irradiation affects nanopar-ticle penetration is important for evaluating the risk of photoagingon topical administration, and also for designing feasible deliveryformulations. Further study is aimed to evaluate the potential ther-apeutic activity of RA-loaded nanoparticles compared to conven-tional RA formulations.

Acknowledgement

The authors are grateful to the financial support ofMinistry of Science and Technology, Taiwan (Grant number:102-2320-B-255-002-MY3).

References

[1] M.L. Etheridge, S.A. Campbell, A.G. Erdman, C.L. Haynes, S.M. Wolf, J.McCullough, The big picture on nanomedicine: the state of investigationaland approved nanomedicine products, Nanomed. Nanotechnol. Biol. Med. 9(2013) 1–14.

[2] T.W. Prow, J.E. Grice, L.L. Lin, R. Faye, M. Butler, W. Becker, E.M.T. Wurm, C.Yoong, T.A. Robertson, H.P. Soyer, M.S. Roberts, Nanoparticles andmicroparticles for skin drug delivery, Adv. Drug Deliv. Rev. 63 (2011) 470–491.

[3] G. Valacchi, C. Sticozzi, A. Pecorelli, F. Cervellati, C. Cervellati, E. Maioli,Cutaneous responses to environmental stressors, Ann. N. Y. Acad. Sci. 1271(2012) 75–81.

[4] L.J. Mortensen, G. Oberdörster, A.P. Pentland, L.A. DeLouise, In vivo skinpenetration of quantum dot nanoparticles in the murine model: the effect ofUVR, Nano Lett. 8 (2008) 2779–2787.

[5] N.A. Monteiro-Riviere, K. Wiench, R. Landsiedel, S. Schulte, A.O. Inman, J.E.Riviere, Safety evaluation of sunscreen formulations containing titaniumdioxide and zinc oxide nanoparticles in UVB sunburned skin: an in vitro andin vivo study, Toxicol. Sci. 123 (2011) 264–280.

[6] L.J. Mortensen, S. Jatana, R. Gelein, A. De Benedetto, K.L. De Mesy Bentley, L.A.Beck, A. Elder, L.A. Delouise, Quantification of quantum dot murine skinpenetration with UVR barrier impairment, Nanotoxicology 7 (2013) 1386–1398.

[7] G.D. Souto, A.R. Pohlmann, S.S. Guterres, Ultraviolet A irradiation increases thepermeation of fullerenes into human and porcine skin from C60-poly(vinylpyrrolidone) aggregate dispersions, Skin Pharmacol. Physiol. 28(2015) 22–30.

[8] D. Papakostas, F. Rancan, W. Sterry, U. Blume-Peytavi, A. Vogt, Nanoparticles indermatology, Arch. Dermatol. Res. 303 (2011) 533–550.

[9] C.L. Fang, S.A. Al-Suwayeh, J.Y. Fang, Nanostructured lipid carriers (NLCs) fordrug delivery and targeting, Recent Pat. Nanotechnol. 7 (2013) 41–55.

[10] F. Danhier, E. Ansorena, J.M. Silva, R. Coco, A. Le Breton, V. Préat, PLGA-basednanoparticles: an overview of biomedical applications, J. Control. Release 161(2012) 505–522.

[11] A.F. Ourique, A. Melero, C. de Bona, U.F. da Silva, A.R. Schaefer, S.S. Pohlmann,C.M. Guterres, K.H. Lehr, C.R.Beck. Kostka, Improved photostability andreduced skin permeation of tretinoin: development of a semisolidnanomedicine, Eur. J. Pharm. Biopharm. 79 (2011) 95–101.

[12] R. Darlenski, C. Surber, J.W. Fluhr, Topical retinoids in the management ofphotodamaged skin: from theory to evidence-based practical approach, Br. J.Dermatol. 163 (2010) 1157–1165.

[13] H.E. Baldwin, M. Nighland, C. Kendall, D.A. Mays, R. Grossman, J. Newburger,40 years of topical tretinoin use in review, J. Drugs Dermatol. 12 (2013) 638–642.

[14] C.H. Lin, Y.P. Fang, S.A. Al-Suwayeh, S.Y. Yang, J.Y. Fang, Percutaneousabsorption and antibacterial activities of lipid nanocarriers loaded with dualdrugs for acne treatment, Biol. Pharm. Bull. 36 (2013) 276–286.

[15] T.L. Pan, P.W. Wang, W.R. Lee, C.L. Fang, C.C. Chen, C.M. Huang, J.Y. Fang,Systematic evaluations of skin damage irradiated by an erbium:YAG laser:histopathologic analysis, proteomic profiles, and cellular response, J. Dermatol.Sci. 58 (2010) 8–18.

[16] C.C. Huang, J.Y. Fang, W.B. Wu, H.S. Chiang, Y.J. Wei, C.F. Hung, Protectiveeffects of (-)-epicatechin-3-gallate on UVA-induced damage in HaCaTkeratinocytes, Arch. Dermatol. Res. 296 (2005) 473–481.

[17] C.J. Wen, T.C. Yen, S.A. Al-Suwayeh, H.W. Chang, J.Y. Fang, In vivo real-timefluorescence visualization and brain-targeting mechanisms of lipidnanocarriers with different fatty ester:oil ratios, Nanomedicine 6 (2011)1545–1559.

[18] J.M. Cho, Y.H. Lee, R.M. Baek, S.W. Lee, Effect of platelet-rich plasma onultraviolet B-induced skin wrinkles in nude mice, J. Plast. Reconst. Aesthet.Surg. 64 (2011) e31–e39.

[19] V. Staniforth, W.C. Huang, K. Aravindaram, N.S. Yang, Ferulic acid, a phenolicphytochemical, inhibits UVB-induced matrix metalloproteinases in mouseskin via posttranslational mechanisms, J. Nutr. Biochem. 23 (2012) 443–451.

[20] W.R. Lee, S.C. Shen, I.A. Aljuffali, Y.C. Li, J.Y. Fang, Erbium-yttrium-aluminum-garnet laser irradiation ameliorates skin permeation and the follicular deliveryof antialopecia drugs, J. Pharm. Sci. 103 (2014) 3542–3552.

[21] F. Rancan, D. Papakostas, S. Hadam, S. Hackbarth, T. Delair, C. Primard, B.Verrier, W. Sterry, U. Blume-Peytavi, A. Vogt, Investigation of polylactic acid(PLA) nanoparticles as drug delivery systems for local dermatotherapy, Pharm.Res. 26 (2009) 2027–2036.

[22] G.A. Castro, C.A. Oliveira, G.A. Mahecha, L.A. Ferreira, Comedolytic effect andreduced skin irritation of a new formulation of all-trans retinoic acid-loadedsolid lipid nanoparticles for topical treatment of acne, Arch. Dermatol. Res. 303(2011) 513–520.

[23] S. Gattu, H.I. Maibach, Enhanced absorption through damaged skin: anoverview of the in vitro human model, Skin Pharmacol. Physiol. 23 (2010)171–176.

[24] A. Horatake, Y. Uchida, M. Schmuth, O. Tanno, R. Yasuda, J.H. Epstein, P.M.Elias, W.M. Holleran, UVB-induced alterations in permeability barrierfunction: roles for epidermal hyperproliferation and thymocyte-mediatedresponse, J. Invest. Dermatol. 108 (1997) 769–775.

[25] K. Tsukahara, S. Moriwaki, M. Hotta, T. Fujimura, Y. Sugiyama-Nakagiri, S.Sugawara, T. Kitahara, Y. Takema, The effect of sunscreen on skin elastaseactivity induced by ultraviolet-A irradiation, Biol. Pharm. Bull. 28 (2005)2302–2307.

[26] M. Ramos-e-Silva, C.M.C. Jacques, Epidermal barrier function and systemicdiseases, Clin. Dermatol. 30 (2012) 277–279.

[27] A. Ishida-Yamamoto, S. Igawa, Genetic skin diseases related to desmosomesand corneodesmosomes, J. Dermatol. Sci. 74 (2014) 99–105.

[28] C.F. Hung, C.L. Fang, S.A. Al-Suwayeh, S.Y. Yang, J.Y. Fang, Evaluation of drugand sunscreen permeation via skin irradiated with UVA and UVB: comparisonsof normal skin and chronologically aged skin, J. Dermatol. Sci. 68 (2012) 135–148.

[29] M. Manconi, C. Sinico, C. Caddeo, A.O. Vila, D. Valenti, A.M. Fadda, Penetrationenhancer containing vesicles as carriers for dermal delivery of tretinoin, Int. J.Pharm. 412 (2011) 37–46.

[30] M. Lapteva, K. Mondon, M. Möller, R. Gurny, Y.N. Kalia, Polymeric micellenanocarriers for the cutaneous delivery of tacrolimus: a targeted approach forthe treatment of psoriasis, Mol. Pharm. 11 (2014) 2898–3001.

[31] H. Zhai, H.I. Maibach, Occlusion vs. skin barrier function, Skin Res. Technol. 8(2002) 1–6.

[32] M.M.A. Abdel-Mottaleb, D. Neumann, A. Lamprecht, Lipid nanocapsules fordermal application: a comparative study of lipid-based versus polymer-basednanocarriers, Eur. J. Pharm. Biopharm. 79 (2001) 36–42.

[33] M. Schneider, F. Stracke, S. Hansen, U.F. Schaefer, Nanoparticles and theirinteractions with the dermal barrier, Dermato-Endocrinology 1 (2009) 197–206.

[34] C.F. Hung, W.Y. Chen, I.A. Aljuffali, H.C. Shih, J.Y. Fang, The risk ofhydroquinone and sunscreen over-absorption via photodamaged skin is notgreater in senescent skin as compared to young skin: nude mouse as an animalmodel, Int. J. Pharm. 471 (2014) 135–145.

[35] K. Biniek, K. Levi, R.H. Dauskardt, Solar UV radiation reduces the barrierfunction of human skin, Proc. Natl. Acad. Sci. USA 109 (2012) 17111–17116.

[36] J.E. Grice, S. Ciotti, N. Weiner, P. Lockwood, S.E. Cross, M.S. Roberts, Relativeuptake of minoxidil into appendages and stratum corneum and permeationthrough human skin in vitro, J. Pharm. Sci. 99 (2010) 712–718.

[37] C.L. Fang, I.A. Aljuffali, Y.C. Li, J.Y. Fang, Delivery and targeting of nanoparticlesinto hair follicles, Ther. Deliv. 5 (2014) 991–1006.

[38] M. Morgen, G.W. Lu, D. Du, R. Stehle, F. Lembke, J. Cervantes, S. Ciotti, R.Haskell, D. Smithey, K. Haley, C. Fan, Targeted delivery of a poorly water-soluble compound to hair follicles using polymeric nanoparticle suspensions,Int. J. Pharm. 416 (2011) 314–322.

[39] S.Y. Jeon, L.Q. Pi, W.S. Lee, Comparison of hair shaft damage after UVA and UVBirradiation, J. Cosmet. Sci. 59 (2008) 151–156.

[40] E. Główka, H. Wosicka-Frackowiak, K. Hyla, J. Stefanowska, K. Jastrzebska, Ł.Klapiszewski, T. Jesionowski, K. Cal, Polymeric nanoparticles-embeddedorganogel for roxithromycin delivery to hair follicles, Eur. J. Pharm.Biopharm. 88 (2014) 75–84.

[41] B. Baroli, Penetration of nanoparticles and nanomaterials in the skin: fiction orreality?, J Pharm. Sci. 99 (2010) 21–50.

[42] B. Baroli, Skin absorption and potential toxicity of nanoparticulatenanomaterials, J. Biomed. Nanotechnol. 6 (2010) 485–496.

[43] P. Charoenputtakun, B. Pamornpathomkul, P. Opanasopit, T. Rojanarata, T.Ngawhirunpat, Terpene composited lipid nanoparticles for enhanced dermaldelivery of all-trans-retinoic acids, Biol. Pharm. Bull. 37 (2014) 1139–1148.

[44] J. Pardeike, A. Hommoss, R.H. Müller, Lipid nanoparticles (SLN, NLC) incosmetic and pharmaceutical dermal products, Int. J. Pharm. 366 (2009) 170–184.

[45] A. Kumar, S.K. Yadav, S.C. Yadav, Biodegradable polymeric nanoparticles baseddrug delivery systems, Colloids Surf. B 75 (2010) 1–18.

[46] P.L. Honeywell-Nguyen, G.S. Gooris, J.A. Bouwstra, Quantitative assessment ofthe transport of elastic and rigid vesicle components and a model drug fromthese vesicle formulations into human skin in vivo, J. Invest. Dermatol. 123(2004) 902–910.

C.-F. Hung et al. / European Journal of Pharmaceutics and Biopharmaceutics 94 (2015) 94–105 105

[47] F. Rancan, Q. Gao, C. Graf, S. Troppens, S. Hadam, S. Hackbarth, C. Kembuan, U.Blume-Peytavi, E. Rühl, J. Lademann, A. Vogt, Skin penetration and cellularuptake of amorphous silica nanoparticles with variable size, surfacefunctionalization, and colloidal stability, ACS Nano 6 (2012) 6829–6842.

[48] N. Provost, M. Moreau, A. Leturque, C. Nizard, Ultraviolet A radiationtransiently disrupts gap junctional communication in human keratinocytes,Am. J. Physiol. Cell Physiol. 284 (2003) C51–C59.

[49] P.S. Peres, V.A. Terra, F.A. Guarnier, R. Cecchini, A.L. Cecchini, Photoaging andchronological aging profile: understanding oxidation of the skin, J. Photochem.Photobiol. B 103 (2011) 93–97.

[50] F. Alexis, E. Pridgen, L.K. Molnar, O.C. Farokhzad, Factors affecting the clearanceand biodistribution of polymeric nanoparticles, Mol. Pharm. 5 (2008) 505–515.

[51] Z. Su, J. Niu, Y. Xiao, Q. Ping, M. Sun, A. Huang, W. You, X. Sang, D. Yuan, Effectof octreotide-polyethylene glycol(100) monostearate modification on thepharmacokinetics and cellular uptake of nanostructured lipid carrier loadedwith hydroxycamptothecine, Mol. Pharm. 8 (2011) 1641–1651.

[52] M. Benfer, T. Kissel, Cell uptake mechanism and knockdown activity of siRNA-loaded biodegradable DEAPA-PVA-g-PLGA nanoparticles, Eur. J. Pharm.Biopharm. 80 (2012) 247–256.

[53] S. Kato, R. Kikuchi, H. Aoshima, Y. Saitoh, N. Miwa, Defensive effects offullerene-C60/liposome complex against UVA-induced intracellular reactiveoxygen species generation and cell death in human skin keratinocytes HaCaT,associated with intracellular uptake and extracellular excretion of fullerene-C60, J. Photochem. Photobiol., B 98 (2010) 144–151.

[54] L.J. Mortensen, S. Ravichandran, L.A. DeLouise, The impact of UVB exposureand differentiation state of primary keratinocytes on their interaction withquantum dots, Nanotoxicology 7 (2013) 1244–1254.

[55] V.A. De Leo, H. Horlick, D. Hanson, M. Eisinger, L.C. Harber, Ultravioletradiation induces changes in membrane metabolism of human keratinocytesin culture, J. Invest. Dermatol. 83 (1984) 323–326.