Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321 – 1330 nanomedjournal.com

Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells Ibrahim A. Aljuffali, PhD a , Tai-Long Pan, PhD b, c , Calvin T. Sung, BSc d , Shu-Hao Chang, MSc e , Jia-You Fang, PhD e, f, g,⁎ a

Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia b School of Traditional Chinese Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan c Liver Research Center, Division of Hepatology, Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan d Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, USA e Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan f Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan g Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan Received 8 December 2014; accepted 14 April 2015

Abstract This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determine whether targeted Minoxidil (MXD) delivery to the follicles and dermal papilla cells (DPCs) could be achieved. Squalene and hexadecyl palmitate (HP) were used as the matrix of the squarticles. The PDGF-squarticles showed a mean diameter and zeta potential of 195 nm and − 46 mV, respectively. Nanoparticle encapsulation enhanced MXD porcine skin deposition from 0.11 to 0.23 μg/mg. The antibody-conjugated nanoparticles ameliorated follicular uptake of MXD by 3-fold compared to that of the control solution in the in vivo mouse model. Both vertical and horizontal skin sections exhibited a wide distribution of nanoparticles in the follicles, epidermis, and deeper skin strata. The encapsulated MXD moderately elicited proliferation of DPCs and vascular endothelial growth factor (VEGF) expression. The active targeting of PDGF-squarticles may be advantageous to improving the limited success of alopecia therapy. © 2015 Elsevier Inc. All rights reserved. Key words: Alopecia; Minoxidil; Squarticles; Hair follicle; Dermal papilla cells

Introduction Androgenetic alopecia (AGA) is one of the most common chronic problems in dermatology caused by the progressive loss

Abbreviations: AFM, atomic force microscopy; AGA, androgenetic alopecia; DPCs, dermal papilla cells; DSC, differential scanning calorimetry; EDC, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide; H&E, hematoxylin and eosin; HP, hexadecyl palmitate; HPLC, high-performance liquid chromatography; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MW, molecular weight; MXD, minoxidil; PDGF, plateletderived growth factor; PG, propylene glycol; SPF, specific pathogen-free; VEGF, vascular endothelial growth factor. There is no disclosure. We have no conflict of interest. ⁎Corresponding author at: Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan 333, Taiwan. E-mail address: [email protected] (J.-Y. Fang). http://dx.doi.org/10.1016/j.nano.2015.04.009 1549-9634/© 2015 Elsevier Inc. All rights reserved.

and thinning of the scalp hair. Despite the high prevalence of alopecia, there are limited options for approved drug treatment. 1 Topical application of 5% minoxidil (MXD) is currently the first-line therapy for AGA. 2 This drug directly induces proliferation of the dermal papilla cells (DPCs) and exerts a vasodilator effect on the hair follicles. 3 However, a recommended twice-daily administration may produce adverse reactions such as scalp dryness, burning, redness, and contact dermatitis. Alcohol and propylene glycol (PG) commonly employed in the commercial products of MXD also cause skin irritation. 4 A long-term application (6-8 weeks) is needed to see improvement in hair loss. 5 It is therefore essential to find an efficient way to enhance drug delivery into targeted lesions and minimize distribution in normal tissues. Nanocarriers provide a strategy for delivering the drugs to the hair follicles. Recently we introduced “squarticles,” nanostructured lipid carriers (NLCs) containing squalene and Precirol® as the lipid cores, for promoting drug uptake in the follicles. 6

1322

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

Squarticles largely accumulate in the follicles due to the interaction with sebum, which is rich in squalene. Although the employment of nanoparticles can increase drug delivery into the follicles, triggering pharmacological activity is of no use if the drug cannot reach the targeted cells. To achieve active targeting to the selected cells, antibody–nanoparticle conjugates are designed to bind the cells via overexpressed antigens on the membrane. The main targets of MXD are DPCs, which are regional stem cells regulating hair development and growth. 7 Previous studies 8,9 have found that DPCs express plateletderived growth factor (PDGF) receptor β. This receptor can be a promising therapeutic target for MXD-loaded nanoparticles. We aimed to design squarticles with a targeting moiety of anti-PDGF receptor β antibody toward DPCs. To this end, in vitro and in vivo experiments for improving the targeted capability of PDGF-squarticles to the hair follicles and DPCs were performed. Sebum consists of wax esters with a percentage of ~ 25%. 10 In this study, hexadecyl palmitate (HP) was used as the solid lipid of squarticles to replace Precirol® employed in our previous work. 6 It is hypothesized that HP, a waxlike substance, further increases the interaction of squarticles with the sebum in the follicles. Both porcine and nude mouse skins were utilized as the permeation barriers in this report. Porcine skin is a good permeation model due to its close resemblance to human skin. 11 The scalp skin appears to be more permeable than the other anatomic sites, especially in alopecia patients. 12 Nude mouse skin offered a skin model with less barrier function.

Methods Preparation of squarticles

Fluorescence microscopy A goat anti-mouse fluorescein isothiocyanate-labeled secondary antibody was used to examine the presence of antibody on the shell of the nanoparticles. The secondary antibody and PDGF-squarticles were mixed for 1 h at a volume ratio of 9:1. The mixture was then centrifuged at 10,000 rpm for 15 min. The excess secondary antibody was removed by washing with PBS three times. The localization of the antibody was monitored using fluorescence microscopy. Average diameter and zeta potential The mean diameter (z-average) and zeta potential of the particles in the nanosystems were measured using a laser-scattering method (Nano ZS90, Malvern). The dispersion was diluted 100-fold with water before testing. Encapsulation efficiency of MXD The percentage of MXD loading in the nanoparticles was measured using an ultracentrifugation method. The dispersion was centrifuged at 48,000 × g and 4 °C for 30 min. The supernatant and precipitate were withdrawn and analyzed using HPLC to calculate the encapsulation percentage of the initial amount of MXD added. 6 Transmission electron microscopy (TEM) One drop of the dispersion was pipetted onto a carbon-filmcoated copper grid to form a thin-film specimen and stained using 1% phosphotungstic acid. The prepared samples were photographed by TEM (H-7500, Hitachi).

The aqueous phase of the squarticles consisted of water (85.4% of the final product), Pluronic F68 (3.5%), and deoxycholic acid (1.0%). The lipid phase consisted of squalene (2.0%), HP (6.0%), SPC (1.5%), and MXD (0.6%). The two phases were separately heated to 85 °C for 15 min. The aqueous phase was added into the lipid phase and homogenized at 12,000 rpm for 20 min. A probe-type sonicator set at a power of 35 W was used to further mix the dispersions for 15 min.

Atomic force microscopy (AFM)

Preparation of PDGF-squarticles

Differential scanning calorimetry (DSC)

Squarticles (1 ml) were incubated with 50 μl of 40 μg/ml EDC and 10 μl of 100 μg/ml antibody for 4 h at room temperature with shaking. The excess coupling agent and the by-products were removed by centrifugation at 10,000 rpm, and the sediment was washed with PBS (1 ml) three times.

The nanoparticles were freeze-dried before the measurements were taken. The DSC analysis was carried out using a Q2000 calorimeter (TA Instruments). The lyophilized powder was weighed in aluminum pans. The thermal analysis profile was recorded at 0-80 °C at a scan rate of 10 °C/min under nitrogen.

Silver staining

Viability and vascular endothelial growth factor (VEGF) of DPCs

To confirm the conjugation between nanoparticles and the antibody, a silver-staining technique was employed. Briefly, the protein was separated by 8% SDS-PAGE. The loading volume of the samples was 15 μl. Electrophoresis was performed on the gels at 100 V for 1 h. The gel was then fixed with 30% methanol for 15 min and was then washed with water, followed by the addition of sodium thiosulfate (0.8 mM). After being washing with water, the gel was equilibrated with 0.2% silver nitrate for 25 min.

Human hair DPCs were purchased from ScienCell. The viability and VEGF amount of DPCs after treatment of nanoparticles were modified according to the methods described earlier. 6 In brief, the nanosystems were added into the cultured cells (6 × 10 5 cells/ml) for a 72-h incubation at 37 °C. The dose of MXD was set at 1 μM in the cell medium. For the VEGF assay, a human VEGF ELISA kit (Invitrogen, Camarillo, CA, USA) was used to examine the VEGF amount in both the

AFM (XE-7, Park Systems) was used to view the morphology and surface state of the nanoparticles. A drop of the dispersion was deposited on a glass slide and left to dry. The observation was operated in a non-contact mode by PointProbe® Plus silicon tips (Nanosensors, Neuchatel). The length and radius of the tip were 125 μm and 7 nm, respectively.

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

centrifuged supernatant and cell extract after a 72-h treatment with nanoparticles. Nanoparticle internalization into DPCs The intracellular uptake of nanoparticles in DPCs was detected by flow cytometry and fluorescence microscopy. DPCs (1 × 10 4 cells/ml) were equilibrated at 37 °C for 24 h and subsequently treated with nanoparticles (15 μl) containing rhodamine 800 (0.1 mg/ml) for 4 h. After washing with PBS twice, a 200-μl trypsin was incubated for 5 min. A 200-μl culture medium was added to stop the reaction. The fluorescence of rhodamine 800 in the cells was determined by flow cytometry. The cells were also subjected to monitoring of the nanoparticle internalization by fluorescence microscopy. 11 Animals Specific pathogen-free (SPF) pigs (1 week old) were provided by the Agricultural Technology Research Institute (Miaoli, Taiwan). Female nude mice (8 weeks old) were supplied by the National Laboratory Animal Center (Taipei, Taiwan). This study was carried out in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of Chang Gung University of Science and Technology. Ethical issues with animal experiments complied with Directive 86/109/EEC from the European Commission. The protocol was approved by the Institutional Animal Care and Use Committee of Chang Gung University of Science and Technology (Permit Number: 2013-005). In vitro skin permeation The in vitro absorption was conducted using Franz diffusion cells. The cellulose membrane, intact skin, or sebum-removed skin was mounted between the donor and the receptor compartments with the stratum corneum (SC) facing up toward the donor. The sebum-removed skin was prepared by washing the skin surface with n-hexane for 5 min. The other procedures and skin extraction method of in vitro MXD absorption were the same as in our previous study. 13 In vivo skin permeation A glass cylinder with a hollow area of 0.785 cm 2 was attached to the mouse’s back using superglue. An aliquot of 0.2 ml control solution or nanosystems containing 0.1 mg/ml Nile red as the fluorescence dye was pipetted into the cylinder. The application period was 4 h. The animal was then sacrificed, and the tested skin area was excised to assess the fluorescence distribution in the skin using vertical and horizontal observations. For vertical observation, the skin was sectioned in a cryostat microtome at a thickness of 10 μm. The slices were viewed using an inverted microscope. For horizontal observation of the skin, the full-thickness skin was examined using confocal microscopy (TCS SP2, Leica). The thickness of the skin was scanned at 5 μm increments via z-axis. The fluorescence within the skin was imaged by summing 15 fragments at various depths from the skin surface.

1323

In vivo hair follicle uptake Differential stripping and cyanoacrylate skin surface casting were utilized to detect the MXD content in the follicles as previously described. 13 Statistical analysis Statistical analysis of the differences between the groups was performed using the Kruskal–Wallis test. The post hoc test for checking individual differences was Dunn’s test. A 0.05 level of probability was taken as the level of significance. Results Physicochemical characterization of the nanoparticles The electrophoresis profiles in Figure 1, A show a band for the anti-PDGF receptor antibody with a molecular weight (MW) of ~ 130 kDa. This value approximated the MW described in the manufacturer’s guide (124 kDa). The band of PDGF-squarticles became less mobile in the electric field than the unbound antibody, suggesting the successful covalent binding between the antibody and the nanoparticles. The attachment of the antibody to the nanoparticulate surface was also checked by fluorescence microscopy using the FITC-labeled antibody. Unlike the squarticles without antibody, PDGF-squarticles showed green fluorescence under the microscope as illustrated in Figure 1, B. These results confirmed the immobilization of the antibody on the surface of the PDGF-squarticles. As shown in Table 1, squarticles without antibody exhibited a mean diameter of 236 nm. The inclusion of PDGF antibody caused a reduction in particulate size (195 nm). The polydispersity (PDI) was 0.29 and 0.19 for squarticles and PDGF-squarticles, respectively, suggesting a narrow size distribution. The zeta potential of the squarticles was anionic (− 44 mV) due to the presence of deoxycholic acid. The antibody conjugation did not affect the surface charge of the nanoparticles (P N 0.05). Both nanocarriers displayed an MXD loading capacity of ~ 50%. Figure 2, A is the TEM of both nanoparticles with different magnifications. The morphology of the nanoparticles was spherical and intact. The particulate size measured by TEM was similar to that measured by laser-scattering. The upper panel of Figure 2, B depicts the topographic image of AFM. In the three-dimensional map of both nanosystems, we found that these particles had the shape of a sphere or oval. We used a phase-imaging mode to map the variations in the surface properties between the squarticles and the PDGF-squarticles. This mode refers to the monitoring of the phase lag between the signals that drive cantilever oscillation and the output signals. The change in the phase lag corresponds to the change of the surface properties. The lower panel of Figure 2, B reveals the images of nanoparticles using the phase-imaging mode. Surface functionalization of the nanoparticles by the antibody significantly changed the phase image. The crystalline characteristics of the nanoparticulate matrix were examined by DSC. Bulk HP showed a sharp peak at 55.3 °C. The melting peak of HP in the squarticles decreased to 51.3 °C. The signal was further reduced to 51.0 °C by antibody incorporation. The melting enthalpy

1324

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

Figure 1. The confirmation of conjugation between anti-PDGF receptor β antibody and squarticles: (A) the silver staining photograph and (B) the fluorescence imaging of squarticles and PDGF-squarticles after a conjugation with goat anti-mouse fluorescein isothiocyanate-labeled secondary antibody.

Table 1 The characterization of the squarticles and PDGF-squarticles by particle size, polydispersity index (PDI), zeta potential, and minoxidil encapsulation percentage. Formulation

Size (nm)

PDI

Zeta potential (mV)

Encapsulation percentage (%)

Squarticles 236.0 ± 3.3 0.29 ± 0.03 − 43.8 ± 0.9 54.4 ± 1.8 PDGF-squarticles 194.5 ± 4.7 0.19 ± 0.01 − 45.5 ± 0.6 49.2 ± 1.2 Each value represents the mean ± SD (n = 3).

exhibited a trend of PDGF-squarticles (100.9 J/g) b squarticles (122.7 J/g) b bulk HP (236.7 J/g). This indicated a higher degree of crystalline disorder by antibody conjugation on the particulate surface. Nanoparticle targeting to DPCs The human DPCs were treated with the MXD-loaded nanocarriers to examine the ability to increase the proliferation as shown in Figure 3, A. PDGF-squarticles elevated cell viability to N 140% as compared to the nontreated control. This effect was significantly greater (P b 0.05) than that achieved by solution and squarticles. The increase in extracellular VEGF expression was detected in the DPCs treated by MXD in solution and nanoparticles (left panel of Figure 3, B). The trend toward

increased VEGF correlated with that of cell proliferation (control b solution b squarticles b PDGF-squarticles). The intracellular VEGF level was comparable (P N 0.05) for the three MXD formulations as shown in the right panel of Figure 3, B. As shown in Figure 3, C, the cells were associated with squarticles as a clear rightward shift of the fluorescence in the histogram compared to the control group. This indicates that the nanoparticles without antibody were sufficient to be internalized by DPCs. The conjugated antibody further shifted the fluorescence signal, demonstrating a potent binding affinity to the PDGF receptors of DPCs. Figure 3, D represents the fluorescence image of DPCs after treatment of the nanoparticles containing fluorescent dye. No red fluorescence was seen inside the cells of the control group. A weak fluorescence in the cytoplasm was observed after a 4-h treatment with squarticles. The image corroborates a strong affinity between PDGF-squarticles and the cells for the subsequent intracellular uptake. In vitro skin permeation Figure 4, A displays the difference in the release rate of MXD from formulations across the cellulose membrane. The kinetic curve of the control solution revealed an initial increase; then a plateau was reached after 12 h. Nanoparticulate inclusion was able to lower MXD release, with the nanocarriers containing antibody showing the slowest rate. The squarticles’ release

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

1325

Figure 2. The physicochemical properties of squarticles and PDGF-squarticles: (A) transmission electron microscopy (TEM) imaging, (B) atomic force microscopy (AFM) imaging, and (C) differential scanning calorimetry (DSC) curves.

behavior exhibited a biphasic pattern with a burst release in the first 8 h followed by a slower release. A zero-order kinetic could describe the release behavior of the PDGF-squarticles, indicating a sustained and continuous release. When the porcine skin was used as the barrier, the nanoparticles delivered MXD more easily than PG solution (Figure 4, B). Both nanosystems showed a 2.5-fold higher flux than did the solution as summarized in Table 2. A similar result was observed by using nude mouse skin as the barrier (Figure 4, C). In vitro MXD deposition in the skin reservoir was measured as shown in Figure 4, D. Encapsulating MXD in the squarticles and PDGF-squarticles increased porcine skin accumulation 2.8- and 2.2-fold more than in the case of solution, respectively. With respect to the mouse skin, we observed 4.0 and 8.7 times higher skin deposition as MXD was loaded in the nanoparticles without and with antibody compared to solution. We compared the MXD flux between intact and sebumremoved skin to determine the influence of sebum on nanoparticle permeation. The flux ratio of sebum-removed skin compared to intact skin (ratioSR/intact) was calculated. As shown

in Table 2, removal of sebum leads to a greater ratioSR/intact for solution than for nanoparticles in both porcine and mouse skin. The lack of sebum even increased the drug flux compared to intact porcine skin. This indicates the necessity of sebum for nanoparticles to represent the potency of enhancing MXD permeation. In vivo skin permeation Figure 5, A demonstrates representative bright-field and fluorescence photographs of the skin after in vivo topical application of PG solution and nanoparticles for 4 h. Some fluorescence was observed for the skin without any treatment. The administration of Nile red-loaded solution did not increase the fluorescence signal compared with nontreated skin. The nanoparticle-treated skin showed a substantial fluorescence. The fluorescence was seen within the epidermis and hair follicles. The orange signal was also broadly distributed in the dermis of the skin treated by squarticles. The vertical-view skin images clearly indicated the extensive diffusion of the nanoparticles to the follicles and the deeper skin

1326

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

Figure 3. The effect of squarticles and PDGF-squarticles on human dermal papilla cells: (A) cell viability (%) of human dermal papilla cells, (B) VEGF expression (pg/ml) in supernatant and extract of human dermal papilla cells, (C) flow cytometry profiles, and (D) nanoparticle internalization into human dermal papilla cells examined by fluorescence microscopy. All data represent the mean ± SD of six experiments.

strata. Confocal microscopy was employed to inspect the skin distribution of Nile red using a horizontal view. As shown in Figure 5, B, the summary of the images taken at a different depth from nontreated skin displays very weak fluorescence. Both nanocarriers showed greater fluorescence intensity compared with the solution. The fluorescence from the nanoparticles largely accumulated in the hair shafts. Figure 5, C exhibits the in vivo uptake of MXD in the hair follicles at the 4-h time point. The follicular uptake of MXD could be improved by entrapping into the nanoparticles. PDGF-squarticles revealed the maximum drug uptake (P b 0.05) in the follicles. The follicular deposition of squarticles and PDGF-squarticles showed 2- and 3-fold enhancement compared to that of the solution, respectively. Discussion In this work, we chose the PDGF receptor as a target for MXD-loaded nanoparticles as it was capable of specifically

delivering MXD to the DPCs in the hair follicles. The experimental results demonstrated that PDGF-squarticles are an active targeting nanocarrier that could largely deposit in the follicles and penetrate into the DPCs, thus a moderate proliferation and VEGF release can be expected. Antibodyconjugated nanoparticle interaction or immunization with skin cells, including epidermal keratinocytes, dendritic cells, and Langerhans cells, has been documented in recent years. 14,15 We showed, for the first time, that antibody-based nanoparticle delivery could target DPCs. The antibody intercalation led to the reduction of the particulate diameter, perhaps due to the superior interfacial packing by the antibody. The antibody may exert a nature of surfactant/emulsifier to reduce the interfacial tension, thus leading to the strong cohesion of the nanoparticulate shell. The zeta potential of our developed nanocarriers was more negative than − 40 mV. This value suggests the low incidence of particle aggregation during storage. 16 The encapsulation percentage of

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

1327

Figure 4. In vitro skin permeation of minoxidil from control solution and nanoparticles assessed by Franz diffusion cell: (A) cumulative amount–time profiles across cellulose membrane, (B) cumulative amount–time profiles across porcine skin, (C) cumulative amount–time profiles across nude mouse skin, and (D) skin deposition in porcine and nude mouse skins. All data represent the mean ± SD of four experiments.

Table 2 The comparison of minoxidil flux (μg/cm 2/h) via intact and sebum-removal skins. Skin Porcine

Formulation

Solution Squarticles PDGF-squarticles Nude Solution mouse Squarticles PDGF-squarticles

Sebum-removal RatioSR/intact a

Intact 7.88 19.84 19.97 11.75 14.42 22.06

± ± ± ± ± ±

1.11 2.10 1.68 2.89 3.12 1.80

13.04 14.78 17.35 10.72 11.18 11.83

± ± ± ± ± ±

1.19 0.73 2.39 1.58 0.65 0.70

1.65 0.74 0.87 0.91 0.76 0.54

Each value represents the mean ± SD (n = 4). a RatioSR/intact, the flux ratio between sebum-removal skin and intact skin.

MXD in the nanocarriers was ~ 50%. This loading capacity was not regarded as low since MXD can be categorized as a hydrophilic agent (log P = 1.2). 17 The core inside the nanopar-

ticles showed less crystalline form according to the DSC profiles. The lipid matrix thus could accommodate MXD molecules with sufficient entrapment. The mechanisms of MXD action on DPCs are enhanced cell proliferation and VEGF overexpression. 18 A previous study has suggested that PDGF receptor β is strongly expressed in DPCs within hair follicles. 8 PDGF receptor β signaling is generally recognized as a modulator involved in hematopoiesis and vessel formation. 19 Although the follicular keratinocytes also express PDGF receptor α and β, 8 both types of PDGF receptors are weakly positive in the keratinocytes. Previous studies 20,21 also demonstrated a lack of PDGF receptors in human keratinocytes. We hypothesized that the PDGF-squarticles can specifically bind to DPCs. In addition to the interaction between the receptor and the antibody on the particulate surface, the size of PDGFsquarticles was feasible for receptor-mediated endocytosis since a diameter of 200 nm is the size limit for intracellular uptake. 22 A particle size of ~ 200 nm is suitable for internalization by the

1328

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

Figure 5. In vivo skin permeation of minoxidil from control solution and nanoparticles using nude mouse as the animal model: (A) hematoxylin and eosin staining and fluorescence microscopy imaging of nude mouse skin, (B) confocal micrographs of nude mouse skin, and (C) follicular uptake. The data of follicular uptake represent the mean ± SD of six experiments.

cells through a clathrin-mediated endocytosis. 23,24 The antibody-conjugated nanoparticles targeting some growth factor receptors such as an EGF receptor can easily trigger a clathrin-mediated pathway. 25 It is proposed that PDGFsquarticles may be endocytosed by DPCs via a similar pathway. We had used a 20% PG/buffer as the control solution for percutaneous MXD absorption. A previous study 26 suggested the increase of MXD permeation by PG-rich vehicles. The developed nanocarriers in this study further enhanced MXD delivery. There were some mechanisms explaining the

permeation enhancement by lipid nanosystems. The small size of nanoparticles leads to close contact with the SC. This occlusive effect increases skin hydration and the possibility of drug partitioning from the vehicle to the SC. As demonstrated previously, 27 the skin distribution of polymeric nanoparticles with a diameter of 100 nm is limited on the SC surface and follicles. Our fluorescence and confocal images signified an extensive distribution of squarticles into the epidermis and the deeper skin strata. Polymeric nanoparticles can be classified as rigid particles. On the other hand, lipid nanocarriers exhibited a

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

softer property than polymeric nanoparticles due to their less-crystalline matrix as proved by DSC. The particles with high flexibility easily penetrate into furrows and follicular openings after close contact with the skin surface. 28 Efficient skin delivery is dependent upon the partitioning of the drug from the vehicle to the sebum. 29 According to the data of MXD permeation via sebum-removed skin, MXD in solution showed a limited ability to interact with sebum since the drug penetration was significantly increased in the absence of sebum. This phenomenon could be ameliorated by using squarticles as the drug carriers. Sebum is a mixture of lipids consisting of squalene, wax, glycerols, and fatty acids. The squalene and waxlike HP in the squarticles were helpful in promoting fusion with sebum, increasing drug partitioning into the skin. This effect may be more prominent in alopecia treatment due to the abundant sebum in the scalp region. We had examined follicular drug accumulation in the in vivo status because the excised skin can reduce the follicular reservoir by N 90%. 30 The results demonstrated a limited MXD uptake into the follicles from the PG solution. The sebum in the follicles is a block for aqueous solution since this lubricant basically repels water. 31 Thus MXD in solution had difficulty penetrating through the sebum. The follicles rich in sebum provide a lipophilic environment for the permeants. Lipophilic nanoparticles rather than hydrophilic vehicles can improve follicular delivery. SPC coating is also beneficial for nanoparticle entry into the follicles as verified in the previous study. 32 Because of the lower resistance of the follicular epithelium compared to the perifollicular epithelium, 33 the nanoparticles can diffuse easily and quickly into deeper strata via the follicles. The in vivo skin imaging and in vitro receptor accumulation suggest the ability of lipid nanoparticles to deliver the dye or drug to the lower strata. The in vitro release profiles suggest a slow release of MXD from the nanoparticles, especially the PDGF-squarticles. The antibody targeting and sustained drug release contributed to the more favorable effect of PDGF-squarticles on DPCs. We demonstrated that the lipid nanocarriers containing squalene and HP could carry MXD into the deeper skin strata and hair follicles where DPCs reside. With the help of anti-PDGF receptor β antibody on the particulate surface, the nanoparticles were internalized by DPCs. The antibody-conjugated nanocarriers not only elevated VEGF expression but also elicited cell proliferation. This effect was mild or moderate. Our study presents an active way to target the drug to DPCs. Improved hair growth can be expected following the application of this nanosystem. This is the first report investigating DPC targeting by antibody–nanoparticle conjugates. This study provides a conceivable proof-of-concept for further application in the management of scalp diseases. Once the nanocarriers reach the bottom of follicles, they should pass across the epithelium and basal membrane for targeting to DPCs since the barrier function of follicular epithelium is much lower than the perifollicular epithelium. The evidence of hair growth efficacy of PDGF-squarticles is also important. Further studies are needed and in progress to explore the possible pathways of PDGFsquarticles delivering to DPCs and the hair growth activity of the nanocarriers in animals.

1329

References 1. Varothai S, Bergfeld WF. Androgenetic alopecia: an evidence-based treatment update. Am J Clin Dermatol 2014;15:217-30. 2. Tsuboi R, Itami S, Inui S, Ueki R, Katsuoka K, Kurata S, et al. Guidelines for the management of androgenetic alopecia (2010). J Dermatol 2011;38:1-8. 3. Mura S, Manconi M, Sinico C, Valenti D, Fadda AM. Penetration enhancer-containing vesicles (PEVs) as carriers for cutaneous delivery of monoxidil. Int J Pharm 2009;380:72-9. 4. Padois K, Cantiéni C, Bertholle V, Bardel C, Pirot F, Falson F. Solid lipid nanoparticles suspension versus commercial solutions for dermal delivery of monoxidil. Int J Pharm 2011;416:300-4. 5. Messenger AG, Rundegren J. Minoxidil: mechanisms of action on hair growth. Br J Dermatol 2004;150:186-94. 6. Aljuffali IA, Sung CT, Shen FM, Huang CT, Fang JY. Squarticles as a lipid nanocarrier for delivering diphencyprone and minoxidil to hair follicles and human dermal papilla cells. AAPS J 2014;16:140-50. 7. Kwack MH, Kang BM, Kim MK, Kim JC, Sung YK. Minoxidil activates β-catenin pathway in human dermal papilla cells: a possible explanation for its anagen prolongation effect. J Dermatol Sci 2011;62:154-9. 8. Kamp H, Geilen CC, Sommer C, Blume-Peytavi U. Regulation of PDGF and PDGF receptor in cultured dermal papilla cells and follicular keratinocytes of the human hair follicle. Exp Dermatol 2003;12:662-72. 9. Yu D, Cao Q, He Z, Sun TT. Expression profiles of tyrosine kinases in cultured follicular papilla cells versus dermal fibroblasts. J Invest Dermatol 2004;123:283-90. 10. Huang ZR, Lin YK, Fang JY. Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology. Molecules 2009;14:540-54. 11. Vitorino C, Almeida A, Sousa J, Lamarche I, Gobin P, Marchand S, et al. Passive and active strategies for transdermal delivery using coencapsulating nanostructured lipid carriers: in vitro vs. in vivo studies. Eur J Pharm Biopharm 2014;86:133-44. 12. O’goshi KI, Iguchi M, Tagami H. Functional analysis of the stratum corneum of scalp skin: studies in patients with alopecia areata and androgenetic alopecia. Arch Dermatol Res 2000;292:605-11. 13. Lee WR, Shen SC, Aljuffali IA, Li YC, Fang JY. Erbium-yttriumaluminum-garnet laser irradiation ameliorates skin permeation and the follicular delivery of antialopecia drugs. J Pharm Sci 2014;103:3542-52. 14. Mittal A, Raber AS, Schaefer UF, Weissmann S, Ebensen T, Schulze K, et al. Non-invasive delivery of nanoparticles to hair follicles: a perspective for transcutaneous immunization. Vaccine 2013;31:3442-51. 15. Zhang LW, Monteiro-Riviere NA. Use of confocal microscopy for nanoparticle drug delivery through skin. J Biomed Opt 2013;18:061214. 16. Fan X, Chen J, Shen Q. Docetaxel–nicotinamide complex-loaded nanostructured lipid carriers for transdermal delivery. Int J Pharm 2013;458:296-304. 17. Zhao Y, Brown MB, Jones SA. The effects of particle properties on nanoparticle drug retention and release in dynamic minoxidil foams. Int J Pharm 2010;383:277-84. 18. Alkhalifah A, Alsantali A, Wang E, McElwee KJ, Shapiro J. Alopecia areata update. Part II. Treatment. J Am Acad Dermatol 2010;62:191-202. 19. Chen PH, Chen X, He X. Platelet-derived growth factors and their receptors: structural and functional perspectives. Biochim Biophys Acta 1834;2013:2176-86. 20. Ansel JC, Tiesman JP, Olerud JE, Krueger JF, Tara DC, Shipley GD, et al. Human keratinocytes are a major source of cutaneous platelet-derived growth factor. J Clin Invest 1993;92:671-8. 21. Rollman O, Jensen UB, Ostman A, Bolund L, Gústafsdóttir SM, Jensen TG. Platelet derived growth factor (PDGF) responsive epidermis formed from human keratinocytes transduced with the PDGF beta receptor gene. J Invest Dermatol 2003;120:742-9. 22. Rancan F, Gao Q, Graf C, Troppens S, Hadam S, Hackbarth S, et al. Skin penetration and cellular uptake of amorphous silica nanoparticles with

1330

23. 24.

25. 26.

27.

I.A. Aljuffali et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1321–1330

variable size, surface functionalization, and colloidal stability. ACS Nano 2012;6:6829-42. Fay F, Scott CJ. Antibody-targeted nanoparticles for cancer therapy. Immunotherapy 2011;3:381-94. Abdelghany SM, Schmid D, Deacon J, Jaworski J, Fay F, McLaughlin KM, et al. Enhanced antitumor activity of the photosensitizer meso-tetra(Nmethyl-4-pyridy) porphine tetra tosylate through encapsulation in antibodytargeted chitosan/alginate nanoparticles. Biomacromolecules 2013;14:302-10. Zwang Y, Yarden Y. Systems biology of growth factor-induced receptor endocytosis. Traffic 2009;10:349-63. Grice JE, Ciotti S, Weiner N, Lockwood P, Cross SE, Roberts MS. Relative uptake of minoxidil into appendages and stratum corneum and permeation through human skin in vitro. J Pharm Sci 2010;99:712-8. Morgen M, Lu GW, Du D, Stehle R, Lembke F, Cervantes J, et al. Targeted delivery of a poorly water-soluble compound to hair follicles using polymeric nanoparticle suspensions. Int J Pharm 2011;416:314-22.

28. Jung S, Patzelt A, Otberg N, Thiede G, Sterry W, Lademann J. Strategy of topical vaccination with nanoparticles. J Biomed Opt 2009;14:021001. 29. Główka E, Wosicka-Frąckowiak H, Hyla K, Stefanowska J, Jastrzębska K, Klapiszewski Ł, et al. Polymeric nanoparticles-embedded organogel for roxithromycin delivery to hair follicles. Eur J Pharm Biopharm 2014;88:75-84. 30. Trauer S, Patzelt A, Otberg N, Knorr F, Rozycki C, Balizs G, et al. Permeation of topically applied caffeine through human skin–a comparison of in vivo and in vitro data. Br J Clin Pharmacol 2009;68:181-6. 31. Wosicka H, Cal K. Targeting to the hair follicles: current status and potential. J Dermatol Sci 2010;57:83-9. 32. Raber AS, 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;179:25-32. 33. Rancan F, Papakostas D, Hadam S, Hackbarth S, Delair T, Primard C, et al. Investigation of polylactic acid (PLA) nanoparticles as drug delivery systems for local dermatotherapy. Pharm Res 2009;26:2027-36.

Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells.

This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determi...
2MB Sizes 0 Downloads 22 Views