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Nanoparticle Targeting to Scalp Hair Follicles: New Perspectives for a Topical Therapy for Alopecia Areata

2019; Elsevier BV; Volume: 140; Issue: 1 Linguagem: Inglês

10.1016/j.jid.2019.05.028

ISSN

1523-1747

Autores

Rebekka Christmann, Carla Thomas, Nadine Jager, Anne S. Raber, Brigitta Loretz, Ulrich F. Schaefer, Thomas Tschernig, Thomas Vogt, Claus‐Michael Lehr,

Tópico(s)

Skin and Cellular Biology Research

Resumo

Alopecia areata (AA) is a hair follicle (HF) disorder, in which the immune system attacks the HF and causes reversible hair loss. Even though AA is not a life-threatening disease, an association with psychosocial diseases and a severe drop in quality of life is common (Pratt et al., 2017Pratt C.H. King L.E. Messenger A.G. Christiano A.M. Sundberg J.P. Alopecia areata.Nat Rev Dis Primers. 2017; 3: 17011Crossref PubMed Scopus (172) Google Scholar). There is, as yet, no approved medicine in the therapy for AA. New potent drugs, such as Jak inhibitors, show promising results but incur severe adverse effects (Vogt et al., 2007Vogt A. Hadam S. Heiderhoff M. Audring H. Lademann J. Sterry W. et al.Morphometry of human terminal and vellus hair follicles.Exp Dermatol. 2007; 16: 946-950Crossref PubMed Scopus (69) Google Scholar). For such drugs, targeted delivery to the site of action is essential. The concept for follicular delivery of drug-loaded nanoparticles (NPs) for treatment of hair disorders shows potential. The key benefits of targeted biodegradable polymeric NP delivery into HFs include (i) protection of the encapsulated drug, (ii) minimization of drug exposure to the skin surface, as well as interfollicular permeation, (iii) maximization of the penetration into the HF compared with the free drug (Mathes et al., 2016Mathes C. Melero A. Conrad P. Vogt T. Rigo L. Selzer D. et al.Nanocarriers for optimizing the balance between interfollicular permeation and follicular uptake of topically applied clobetasol to minimize adverse effects.J Control Release. 2016; 223: 207-214Crossref PubMed Scopus (41) Google Scholar), (iv) the possibility of building a drug depot in the upper part of the HF, creating possible protection of the NPs from external influences such as, textile contact, washing (Lademann et al., 2007Lademann J. Richter H. Teichmann A. Otberg N. Blume-Peytavi U. Luengo J. et al.Nanoparticles- an efficient carrier for drug delivery into the hair follicles.Eur J Pharm Biopharm. 2007; 66: 159-164Crossref PubMed Scopus (0) Google Scholar), and (v) the concurrent ability of a sustained drug release from the depot to reduce the application frequency and enhance patient compliance (Hofmeier and Surber, 2017Hofmeier K.S. Surber C. Nanomedicine in dermatology: nanotechnology in prevention, diagnosis, and therapy.in: Müller B. van de Voorde M.H. Nanoscience and nanotechnology for human health. Nanotechnology innovation & applications. Wiley-VCH Press, Weinheim, Germany2017: 329-356Crossref Scopus (2) Google Scholar). Taking this into account, Jak inhibitor-loaded NPs could deposit in the upper part of the HF, release the drug in a controlled manner, which diffuses to the site of action (hair bulb), and be taken up by the follicular epithelial cells and immune cells (Divito and Kupper, 2014Divito S.J. Kupper T.S. Inhibiting Janus kinases to treat alopecia areata.Nat Med. 2014; 20: 989-990Crossref PubMed Scopus (32) Google Scholar); thus reducing adverse effects with less systemic drug and skin exposure. However, a hypothesized penetration mechanism for NP uptake into human HF postulates that, by the movement of the hair shaft, overlapping cuticle cells serve as a gear pump and push the NPs into the HF (Lademann et al., 2007Lademann J. Richter H. Teichmann A. Otberg N. Blume-Peytavi U. Luengo J. et al.Nanoparticles- an efficient carrier for drug delivery into the hair follicles.Eur J Pharm Biopharm. 2007; 66: 159-164Crossref PubMed Scopus (0) Google Scholar, Radtke et al., 2017Radtke M. Patzelt A. Knorr F. Lademann J. Netz R.R. Ratchet effect for nanoparticle transport in hair follicles.Eur J Pharm Biopharm. 2017; 116: 125-130Crossref PubMed Scopus (37) Google Scholar). Additionally, appropriate massage seems to be important in this context (Li et al., 2019Li B.S. Cary J.H. Maibach H.I. Should we instruct patients to rub topical agents into skin?: the evidence.J Dermatolog Treat. 2019; 30: 328-332Crossref PubMed Scopus (4) Google Scholar). Thus, we questioned if NPs are able to penetrate into AA-affected HFs, where there is no hair present. To fill these critical knowledge gaps, we studied NP deposition in AA-affected HFs with a previously developed quantification method for NP uptake into human forearm HFs, demonstrating an excellent in vivo–in vitro correlation with pig ear skin (Raber et al., 2014Raber A.S. Mittal A. Schäfer J. Bakowsky U. Reichrath J. Vogt T. et al.Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.J Control Release. 2014; 179: 25-32Crossref PubMed Scopus (68) Google Scholar). However, there are no quantitative data for human scalp skin. Because hair density and hair type (vellus vs. terminal) differ between the healthy forearm and scalp, such data are critical to assess treatment options. Therefore, we transferred the quantification method of NP uptake into human forearm HF to human scalp skin of healthy volunteers, subsequent transferred it to AA patients. However, the recruitment of participants for such studies is limited. Accordingly, we assessed the quantification method also in the hairy scalp of body donors as a possible alternative to future in vivo studies (experiments were conducted with the skin attached to the corpus; body donors were not fixated before the experiment and kept at 4 °C; maximal post mortem interval was 3 days) (for study group details see Figure 1a). The studies were approved by the Ärztekammer des Saarlandes ethical committee, including informed written consent of participants. For the model NPs, we continued to use the NPs used in the previously developed quantification method for NP uptake into the HF (Raber et al., 2014Raber A.S. Mittal A. Schäfer J. Bakowsky U. Reichrath J. Vogt T. et al.Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.J Control Release. 2014; 179: 25-32Crossref PubMed Scopus (68) Google Scholar). The NPs, size of 150 nm, narrow size distribution and negative zeta potential, consisted of fluorescently covalently labeled poly (lactic-co-glycolic acid) (PLGA), a biodegradable, biocompatible polymer (Wang et al., 2018Wang E.H.C. Sallee B.N. Tejeda C.I. Christiano A.M. JAK inhibitors for treatment of alopecia areata.J Invest Dermatol. 2018; 138: 1911-1916Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, Mittal et al., 2013Mittal A. Raber A.S. Schaefer U.F. Weissmann S. Ebensen T. Schulze K. et al.Non-invasive delivery of nanoparticles to hair follicles: a perspective for transcutaneous immunization.Vaccine. 2013; 31: 3442-3451Crossref PubMed Scopus (51) Google Scholar). For a detailed description of the procedure, see Supplementary Materials and Methods. In brief, differential stripping was proceeded 1 hour after applying 15 μl of aqueous fluorescently covalently labeled PLGA-NP suspension (concentration = 50 mg/ml) per application area by a 3-minute massage (Scientific Committee on Consumer Safety, 2018Scientific Committee on Consumer SafetyThe SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation 10th Revision.https://ec.europa.eu/health/sites/health/files/scientific_committees/consumer_safety/docs/sccs_o_224.pdfDate: 2018Google Scholar), which comprises skin surface cleaning by tape stripping and subsequent removal of HF casts by cyanoacrylate biopsies. For mass balance, all samples in contact with the formulation were extracted in organic solvent and analyzed using fluorescence intensity. Stratum corneum recovery after differential stripping was monitored by transepidermal water loss measurements (Supplementary Figure S1). Experimental sets were excluded if they did not meet the 85–115% mass balance limits as recommended by the Scientific Committee on Consumer Safety guidelines (2018; Figure 1a). Comparing percentage of dose (mean ± SD) of applied NPs recovered on the skin surface (61.0 ± 4.1% vs. 56.4 ± 3.7%), the percentage of dose remaining on the glove (28.1 ± 5.5% vs. 24.1 ± 3.7%), and the percentage left on the skin (0.1 ± 0.2% vs. 1.3 ± 1.1%) after cyanoacrylate biopsies, results for forearm and healthy scalp, respectively, showed no significant differences (Supplementary Table S1; Supplementary Figure S2, red and purple bars), demonstrating successful transfer of the method from forearm to scalp skin. Owing to higher HF density on a healthy scalp, we hypothesized a higher amount of NP deposition in scalp HFs than in forearm HFs. Indeed, the amount of NPs deposited in the HFs per application area of healthy scalp (8.5 ± 3.6% equals 59.79 ± 24.46 μg/HFs per area) was significantly higher than for forearm (2.5 ± 0.9% equals 19.05 ± 12.78 μg/HFs per area) (P ≤ 0.001; Figure 2a, gray bars). To assess the influence of HF density, we documented each application area using a dermatoscope (handyscope, FotoFinder, Bad Birnbach, Germany) (Figure 1b) and determined the number of HF orifices visible on the skin surface of each individual application area manually using ImageJ (ImageJ 1.51j8, NIH). After normalizing the NPs deposited per application area for the different HF orifice densities on the human forearm (0.75 ± 0.57 μg/one HF) and healthy scalp (0.42 ± 0.16 μg/one HF), the differences were no longer statistically significant (Figure 2b), that is, the amount of NPs deposited per HF was about the same, regardless of the body site. Given that this study is limited by its number of participants, the results need to be considered carefully. Nevertheless, this is an interesting result, because vellus and terminal HF show fundamental morphometric differences (Teichmann et al., 2005Teichmann A. Jacobi U. Ossadnik M. Richter H. Koch S. Sterry W. et al.Differential stripping: determination of the amount of topically applied substances penetrated into the hair follicles.J Invest Dermatol. 2005; 125: 264-269Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Also, the HF densities and infundibular surfaces significantly differ between different vellus HF covered body sites (Otberg et al., 2004Otberg N. Richter H. Schaefer H. Blume-Peytavi U. Sterry W. Lademann J. Variations of hair follicle size and distribution in different body sites.J Invest Dermatol. 2004; 122: 14-19Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). This study takes only human forearm HF into account. There was no significant difference when comparing the data of a healthy scalp with a body donation scalp regarding the amount of follicular deposition per application area (59.79 ± 24.46 μg vs. 41.83 ± 18.53 μg) (Figure 2b) or the deposition per single HF (0.42 ± 0.16 μg vs. 0.33 ± 0.21 μg, healthy scalp and body donor scalp, respectively). This implies that body donors may be used as models to develop future follicle-targeted drug carriers for dermatological applications. Finally, we investigated the NP uptake in vivo into AA-affected hairless scalp HFs. Despite the postulated NP uptake mechanism in HF (Lademann et al., 2007Lademann J. Richter H. Teichmann A. Otberg N. Blume-Peytavi U. Luengo J. et al.Nanoparticles- an efficient carrier for drug delivery into the hair follicles.Eur J Pharm Biopharm. 2007; 66: 159-164Crossref PubMed Scopus (0) Google Scholar), we demonstrated NP deposition in human HF in healthy (8.5 ± 3.6% equals 59.79 ± 24.46 μg) and AA-affected HFs (2.7 ± 0.4% equals 20.49 ± 2.90 μg); the difference of NP deposition per application area (Figure 2b, gray bars) between the two groups was significant (P ≤ 0.01). Interestingly, after correction for the number of HF orifices per application area, the NP deposition per single HF (Figure 2b, white bars) was no longer significantly different (P > 0.05; 0.42 ± 0.16 μg vs. 0.23 ± 0.07 μg, healthy vs. AA scalp, respectively). In conclusion, we were able to demonstrate that topically applied NPs do penetrate into human scalp HFs. This phenomenon appeared to be of the same scale as the previously reported follicular penetration in human forearm HFs (Raber et al., 2014Raber A.S. Mittal A. Schäfer J. Bakowsky U. Reichrath J. Vogt T. et al.Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.J Control Release. 2014; 179: 25-32Crossref PubMed Scopus (68) Google Scholar) and thus to be independent of the HF type. Moreover, and, most importantly, NPs were found to penetrate into HFs also in the absence of a hair shaft. This opens possibilities for the treatment of inflammatory hair loss, most importantly AA, by designing appropriate nanomedicines capable of penetrating and delivering their cargo directly to the affected HFs. Datasets related to this article can be found at https://doi.org/10.17632/fn6gg3f4py.1, hosted at Mendeley Data. Rebekka Christmann: http://orcid.org/0000-0003-1775-2113 Carla Thomas: http://orcid.org/0000-0002-9809-9533 Nadine Jager: http://orcid.org/0000-0002-5216-8441 Anne S. Raber: http://orcid.org/0000-0003-0018-2652 Brigitta Loretz: http://orcid.org/0000-0003-0057-5181 Ulrich F. Schaefer: http://orcid.org/0000-0002-2470-2168 Thomas Tschernig: http://orcid.org/0000-0002-7788-1796 Thomas Vogt: http://orcid.org/0000-0002-2824-5005 Claus-Michael Lehr: http://orcid.org/0000-0002-5864-8462 The authors declare no conflict of interest. This work was supported by Dr. Rolf M. Schwiete Stiftung , Mannheim , Germany (Project-Nr. 14/2016). We thank Olga Hartwig for help with confocal laser scanning microscope, UHU, Bühl/Baden, Germany, and tesa SE, Norderstedt, Germany for the kind donation of materials. Conceptualization: TV, UFS, CML; Funding Acquisition: TV, CML; Investigation: RC, CT, NJ; Methodology: ASR, UFS, RC, CT; Project Administration: BL, TV; Resources: TT, CT, TV, RC; Supervision: BL, UFS, CML, TV; Writing - Original Draft Preparation: RC, UFS, BL; Writing - Review and Editing: RC, CT, NJ, ASR, BL, UFS, TT, TV, CML. Initially, hair was cut to 1 mm with an electrical shaver (Braun Bartschneider, BT3040, Procter & Gamble Service, Schwalbach am Taunus, Germany) and the skin surface was cleansed of free hair. Application areas, each 1,767 cm2, were marked with a permanent black marker (Faber-Castell MULTIMARK permanent 152399, A.W. Faber-Castell Vertrieb, Stein, Germany) using a Teflon mask. Each application area was documented using a dermatoscope (handyscope, FotoFinder, Bad Birnbach, Germany) for counting hair follicle (HF) orifices. Then, 15 μl of an aqueous nanoparticle (NP) suspension (concentration = 50 mg/ml) composed of fluoresceinamine covalently labeled poly (lactic-co-glycolic acid) (PLGA) NPs (Mittal et al., 2013Mittal A. Raber A.S. Schaefer U.F. Weissmann S. Ebensen T. Schulze K. et al.Non-invasive delivery of nanoparticles to hair follicles: a perspective for transcutaneous immunization.Vaccine. 2013; 31: 3442-3451Crossref PubMed Scopus (51) Google Scholar, Wang et al., 2018Wang E.H.C. Sallee B.N. Tejeda C.I. Christiano A.M. JAK inhibitors for treatment of alopecia areata.J Invest Dermatol. 2018; 138: 1911-1916Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) was applied per application area. Particle size, 149.04 ± 3.46 nm; polydispersity index, 0.06 ± 0.02; zeta potential −24.64 ± 6.85 mV. On blank areas (one per participant), 15 μl of purified water was applied for individual vehicle control. For NP penetration enhancement into the HF, a 3-minute circular massage with slight pressure (60 r.p.m., ∼2 Newton) was applied by a trained person with a gloved fingertip (Mathes et al., 2016Mathes C. Melero A. Conrad P. Vogt T. Rigo L. Selzer D. et al.Nanocarriers for optimizing the balance between interfollicular permeation and follicular uptake of topically applied clobetasol to minimize adverse effects.J Control Release. 2016; 223: 207-214Crossref PubMed Scopus (41) Google Scholar, Lademann et al., 2007Lademann J. Richter H. Teichmann A. Otberg N. Blume-Peytavi U. Luengo J. et al.Nanoparticles- an efficient carrier for drug delivery into the hair follicles.Eur J Pharm Biopharm. 2007; 66: 159-164Crossref PubMed Scopus (0) Google Scholar). Differential stripping was proceeded after a 1-hour incubation time (Scientific Committee on Consumer Safety, 2018Scientific Committee on Consumer SafetyThe SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation 10th Revision.https://ec.europa.eu/health/sites/health/files/scientific_committees/consumer_safety/docs/sccs_o_224.pdfDate: 2018Google Scholar). First, the skin surface was cleaned by tape stripping, followed by removal of HF casts by cyanoacrylate biopsies. For the tape stripping, the application area was covered by one tape strip (tesafilm kristall-klar, Norderstedt, Germany), pressed onto the skin surface by a paint roller (to reach furrows and crinkles) (Lademann et al., 2007Lademann J. Richter H. Teichmann A. Otberg N. Blume-Peytavi U. Luengo J. et al.Nanoparticles--an efficient carrier for drug delivery into the hair follicles.Eur J Pharm Biopharm. 2007; 66: 159-164Crossref PubMed Scopus (430) Google Scholar), and removed in one swoop with forceps. A total of 12 successive tape strips were used to clean the skin surface by removal of stratum corneum layers. The removal of the HF casts was carried out using the following two cyanoacrylate biopsies. One drop of superglue (UHU blitzschnell Pipette, Bühl/Baden, Germany) was applied to the cleaned surface area and covered by a tape strip. After polymerization of the cyanoacrylate (5 minutes), the tape strip was removed with a forceps in one swoop. This was done twice. It has been shown by Raber et al., 2014Raber A.S. Mittal A. Schäfer J. Bakowsky U. Reichrath J. Vogt T. et al.Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.J Control Release. 2014; 179: 25-32Crossref PubMed Scopus (68) Google Scholar, that with two biopsies a complete removal of the HFs on the application area can be achieved. For mass balance, the treated skin surface and its surroundings were cleaned with a cotton ball. All samples were analyzed separately: glove fingertip, tape strips (one and two separately, then two together), cyanoacrylate biopsies, and cotton ball (skin rest). Extraction and analyzation by fluorescence spectroscopy were done according to Raber et al., 2014Raber A.S. Mittal A. Schäfer J. Bakowsky U. Reichrath J. Vogt T. et al.Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.J Control Release. 2014; 179: 25-32Crossref PubMed Scopus (68) Google Scholar. Relative fluorescence unit values were corrected by the individual blank matrix value. Lower limit of quantification of fluorescently covalently labeled PLGA-NP suspension calibration curve was calculated according to the FDA Bioanalytical Method Validation guideline (Weiss et al., 2006Weiss B. Schaefer U.F. Zapp J. Lamprecht A. Stallmach A. Lehr C.M. Nanoparticles made of fluorescence-labelled Poly(L-lactide-co-glycolide): preparation, stability, and biocompatibility.J Nanosci Nanotechnol. 2006; 6: 3048-3056Crossref PubMed Scopus (0) Google Scholar). Values lower than the lower limit of quantification were excluded. Experimental sets were excluded if they did not meet 85–115% as the recommended range of mass balance by the Scientific Committee on Consumer Safety guidelines (2018). Count of HF orifices on dermatoscopic pictures was done manually by ImageJ (ImageJ 1.51j8, NIH) for each application area individually. By correction of the follicular deposition of NPs per application area to the number of HF orifices counted, the amount of NPs (in μg) deposited into one single HF was determined. Recovery of skin barrier function was monitored by transepidermal water-loss measurements according to the ethical committee (Supplementary Figure S1). DiD-loaded PLGA-NPs were chosen for the visualization of NPs inside the HF, because fluorescently covalently labeled -PLGA-NPs in conjugation with skin and HFs are difficult to differentiate, because of the high auto fluorescence of the organs (Mittal et al., 2013Mittal A. Raber A.S. Schaefer U.F. Weissmann S. Ebensen T. Schulze K. et al.Non-invasive delivery of nanoparticles to hair follicles: a perspective for transcutaneous immunization.Vaccine. 2013; 31: 3442-3451Crossref PubMed Scopus (63) Google Scholar) and the terminated fluorescence intensity of Fluorescein. DiD-loaded PLGA-NPs were prepared as described previously (Mittal et al., 2013Mittal A. Raber A.S. Schaefer U.F. Weissmann S. Ebensen T. Schulze K. et al.Non-invasive delivery of nanoparticles to hair follicles: a perspective for transcutaneous immunization.Vaccine. 2013; 31: 3442-3451Crossref PubMed Scopus (51) Google Scholar) with addition of Vybrant DiD cell-labeling solution (Vybrant Multicolor Cell-Labeling Kit, Invitrogen, Waltham, MA) to the organic polymer solution. The free dye was separated from NPs by dialysis in purified water (Spectra/Por Float-a-Lyzer, 100 kDa, Spectrum Europe B.V., Breda, The Netherlands) for 29 hours. NPs were 124.0 ± 0.9 nm, and had a polydispersity index of 0.07 ± 0.02 and a zeta potential of −21.8 ± 0.2 mV. To visualize the DiD-loaded PLGA-NP deposition inside HFs (before the scalp hair was shortened to 1 mm) per application area (1,767 cm2), 20 μl of the NP formulation (250 mg PLGA/ml) was applied by massage. After the incubation time, no further procedure for skin cleaning (no tape stripping) or hair removal was applied; the applied NP formulation remained on the skin surface and in the HFs. An 8-mm punch biopsy was taken. Blank application areas (Control without NP) were treated the same without application of NP formulation. Subsequently, the skin tissue was imbedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) and cut by cryostat (SLEE, Mainz, Germany) (Supplementary Figure S3). Images were taken with a confocal laser scanning microscope (CLSM) (Leica TCS SP8, Leica, Mannheim, Germany) and processed with Leica Application Suite X (LAS X) software (Leica, Mannheim, Germany). Transversal sections to 100 μm were cut (Supplementary Figure S3c, S3d). On the first section, the amount of NPs on the skin surface was too high for a sufficient differentiation between NPs remaining on the skin surface and deposited in the HF. Therefore, the second 100-μm section is shown in Supplementary Figure S3c, demonstrating the DiD-loaded PLGA-NP (red fluorescence) inside the HF (hair shaft is present) (overlay with transmission light image). On the third 100-μm section, no NPs were detectable (not shown). The second transversal 100-μm section of the blank biopsy is shown in Supplementary Figure S3d (control without NP [HF with hair shaft] [overlay transmission light image and red fluorescence channel). Results of longitudinal tissue sections of 20 μm are shown in Supplementary Figure S3a and b. DiD-loaded PLGA-NPs (red fluorescence) can be detected only on the skin surface and inside the HF (hair shaft is present), counterstaining with DAPI (Sigma, Darmstadt, Germany) (blue fluorescence) (Supplementary Figure S3a, NP in HF). In the blank tissue sections, no strong red fluorescence was detectable (Supplementary Figure S3b, control without NP [HF with hair shaft]) (blue fluorescence: DAPI). Visualization of NPs inside the scalp HFs can only be shown by Body Donors, because ethical reasons do not allow invasive procedures in living participants. In addition, because of ethical reasons, only fluorescently covalently labeled -PLGA-NP for the human in vivo studies could be used.Supplementary Figure S1Transepidermal water loss (TEWL) measurements for skin damage control through differential stripping. Exemplary measurements on healthy forearm and healthy scalp on four participants (after 9 days from one participant). Control measurements were taken on untreated skin (control forearm, white triangle; control scalp, black triangle), Blank measurements on Blank application areas (star) and treated measurements on NP application areas (treated forearm, white square; treated scalp, block square). Results are presented as mean ± SD. Results show slight stratum corneum damage after differential stripping and fast recovery, without permanent skin surface damage by the method. Similar results have been reported by Raber et al., 2014Raber A.S. Mittal A. Schäfer J. Bakowsky U. Reichrath J. Vogt T. et al.Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.J Control Release. 2014; 179: 25-32Crossref PubMed Scopus (68) Google Scholar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Confocal laser scanning microscope (CLSM) images of DiD-loaded PLGA-NPs applied on the scalp of a Body Donor. (a, b) 20-μm longitudinal sections, counterstained with DAPI; left-hand images, no transmission light overlay; right-hand images, with transmission light overlay. (a) CLSM image of DiD-loaded PLGA-NPs (red) in HF (with hair shaft); DiD detection: HeNe 633 laser, emission: 665–680 nm, gain 40; DAPI detection: diode 405 laser, emission: 430–450 nm, gain 60; magnification ×25; pixel size/voxel size: x = 0.455 μm, y = 0.455 μm, z = 2.664 μm; 15 frames. Scale bars = 100 μm. (b) CLSM image of Control without NP in HF (with hair shaft); DiD detection: HeNe 633 laser, emission: 665–680 nm, gain 100; DAPI detection: diode 405 laser, emission: 430– 450 nm, gain 60; magnification ×10; pixel size: x = 0.001 mm, y = 0.001 mm; 1 frame. Scale bars = 200 μm. (c, d) 100-μm transversal sections, overlay with transmission light channel. (c) CLSM image of DiD-loaded PLGA-NPs (red) in HF (with hair shaft); DiD detection: HeNe 633 laser, emission: 665–680 nm, gain 40; magnification ×25; pixel size/voxel size: x = 0.104 μm, y = 0.104 μm, z = −0.568 μm; 137 frames. Scale bar = 100 μm. (d) CLSM image of Control without NP in HF (with hair shaft); DiD detection: HeNe 633 laser, emission: 665–680 nm, gain 40; magnification ×25; pixel size/voxel size: x = 0.455 μm, y = 0.455 μm, z = 3.582 μm; 25 frames. Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Differential stripping data (Mean ± SD) of healthy forearm and healthy scalp, Body Donor scalp and alopecia areata-affected scalpNo. of experimental setsForearmScalpHealthyHealthyBody DonorAlopecia Areata13765% of dose of applied NP per 1,767 cm2 application areaSkin surface61.0 ± 4.156.4 ± 3.765.2 ± 5.560.9 ± 5.4Skin rest0.1±0.21.3 ± 1.10.9 ± 0.70.2 ± 0.20Glove28.1 ± 5.524.1 ± 3.729.3 ± 10.238.7 ± 6.7HF2.5 ± 0.98.5 ± 3.65.4 ± 3.12.7 ± 0.4Mass balance91.7 ± 3.290.3 ± 3.4100.9 ± 3.4102.6 ± 3.1NP deposited in HFs per application area (μg)HF19.05 ± 12.7859.79 ± 24.4641.83 ± 18.5320.49 ± 2.90NP deposited per HF (μg)HF0.75 ± 0.570.42 ± 0.160.33 ± 0.210.23 ± 0.07Boldface indicates deposition in HFs.For statistical analysis, refer to Figure 2b.Abbreviations: HF, hair follicle; NP, nanoparticle. Open table in a new tab Boldface indicates deposition in HFs. For statistical analysis, refer to Figure 2b. Abbreviations: HF, hair follicle; NP, nanoparticle.

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