Enhanced transdermal delivery of curcumin nanosuspensions: A mechanistic study based on co-localization of particle and drug signals

International Journal of Pharmaceutics 588 (2020) 119737

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Enhanced transdermal delivery of curcumin nanosuspensions: A mechanistic study based on co-localization of particle and drug signals

T

Tingting Shia, Yongjiu Lva, Weizi Huanga, Zhezheng Fanga, Jianping Qia, Zhongjian Chenb, ⁎ Weili Zhaoa, Wei Wua, Yi Lua, a b

Key Laboratory of Smart Drug Delivery of MOE, School of Pharmacy, Fudan University, Shanghai 201203, China Shanghai Skin Disease Hospital, Shanghai 200443, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanosuspensions Transdermal delivery Aggregation-caused quenching Hair follicle Curcumin Co-localization

Nanosuspensions have received much attention in enhanced transdermal delivery. However, the corresponding mechanisms have not been clarified. In particular, whether nanosuspensions can directly penetrate across the stratum corneum (SC) and what is the transdermal route for the enhanced penetration. Therefore, curcumin (CUR) was adopted in this study as a model drug, while an aggregation-caused quenching (ACQ) probe was physically embedded in CUR nanosuspensions, i.e., the CUR hybrid nanosuspensions (CUR-HNSs), for bioimaging. The ACQ properties enable identification of intact CUR-HNSs. The co-localization of particle and CUR signals was exploited to outline the translocation profiles of intact nanosuspensions as well as the cargoes. Three sizes of CUR-HNSs are prepared, which are spherical and amorphous. CUR is poor in transdermal transport even in propylene glycol solution, which was enhanced by nanosuspensions. Although 400 nm CUR-HNSs present higher steady state flux than 140 nm and 730 nm ones, the cumulative amount of permeated CUR is yet less than 2% of the applied dose at 12 h. Co-localization of CUR and ACQ probe signals indicates that CUR-HNSs can infiltrate into the SC layer and accumulate in the hair follicles. The intact CUR-HNSs cannot enter into the skin. On the contrary, CUR molecules diffuse into the whole skin tissues following dissolution of CUR-HNSs in the SC and the hair follicles. In conclusion, nanosuspensions are advantageous for transdermal delivery of poorly permeable drugs by filtrate into the SC and accumulate in hair follicles.

1. Introduction Skin is attractive for both local and systemic drug delivery due to the advantages such as avoidance of gastrointestinal irritation and firstpass metabolism, sustained drug release, and good patients’ compliance (Alexander et al., 2012; Hassan and Elshafeey, 2010; Tsai et al., 2014). However, the stratum corneum (SC) forms an entry barrier in the outermost layer of the skin for not only harmful xenobiotics but also most therapeutic compounds (Patzelt et al., 2017; Teichmann et al., 2006). Only small (< 500 Da) and moderately lipophilic molecules (logP 1–3) can diffuse passively across the SC (Giannos, 2015). A myriad of nanovehicles have been developed to improve transdermal or dermal drug delivery. The hypothesis is that the vehicles can penetrate across the SC with the cargos encapsulated. It is yet conflicting whether nanoparticles can permeate into tissues underlying the SC. Some studies indicate that intact vehicles may not enter into the skin but accumulate in the hair follicles, where the cargos are released and diffuse into the skin in the molecular form (Couto et al., 2014; Mak et al., 2011). If that's the case,

⁎

nano-vehicles may be not the optimal carrier in transdermal or dermal drug delivery considering the low drug loading. Nanosuspensions are suspensions of drug nanoparticles with size ranging from dozens to hundreds of nanometers (Lu et al., 2015, 2016; Malamatari et al., 2018; Mohammad et al., 2019b; Xin et al., 2017). Absence of carrier chemicals endows nanosuspensions with high drug loading of 50–100% (w/w), being significantly higher than that of conventional nanoparticles. Besides, the increased specific surface area due to size reduction significantly increases the dissolution of water insoluble drugs, leading to the initial application of nanosuspensions in oral delivery for improved bioavailability (Lu et al., 2016, 2017). Recently, nanosuspensions found new applications in transdermal drug delivery (Mohammad et al., 2019a; Sinico et al., 2017; Vidlářová et al., 2016). The successful marketing of rutin nanosuspensions (Juvedical®) in cosmetic product further encourages research and development in this field (Li et al., 2019; Lu et al., 2017). However, little is known for the enhanced transdermal permeation from nanosuspensions. The proposed mechanisms include: (i) enhanced transdermal concentration

Corresponding author. E-mail address: [email protected] (Y. Lu).

https://doi.org/10.1016/j.ijpharm.2020.119737 Received 15 March 2020; Received in revised form 19 July 2020; Accepted 2 August 2020 Available online 03 August 2020 0378-5173/ © 2020 Elsevier B.V. All rights reserved.

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

2. Materials and methods

gradient due to increased apparent solubility and dissolution; (ii) prolonged retention on skin surface to maintain the concentration gradient (Lu et al., 2016; Sinico et al., 2017). However, provided that the primary role of nanosuspensions is to enhance the concentration gradient for permeation, the significance of this technique for transdermal route is compromised, because a supersaturated drug solution combined with penetration enhancers may be more advantageous (Chen et al., 2020). Pilot studies have been performed to unravel the intradermal fate of nanosuspensions (Corrias et al., 2017; Li et al., 2018; Vidlářová et al., 2016). Scanning electron microscopy (SEM) was used to identify drug nanoparticles in the dermal tissues (Corrias et al., 2017; Li et al., 2018). However, the sample process such as dehydration and dyeing/gold sputtering may confuse drug nanoparticles with backgrounds. Despite excellent resolution, the visual field of SEM is extremely limited, being unable to detect particle transportation through the whole skin layer. Besides, SEM only provides static viewing and lacks the capability for dynamic monitoring. Another strategy depends on auto fluorescent drugs such as curcumin (CUR) and nile red (Corrias et al., 2017; Vidlářová et al., 2016). The intention is to track the transportation of nanosuspensions by the fluorescence of these drugs but fails to discriminate the intact drug particles from dissolved molecules. Conclusions drawn from these results may be misleading. Therefore, the current methods cannot reveal mechanistic information, e.g., “whether and how can nanosuspensions penetrate the skin”. Environment-responsive fluorophores have been exploited to understand the mechanisms of nanocarriers due to the ability of self-discrimination (Chen et al., 2019; Wang et al., 2019; Wu and Li, 2019). Most recently, we find out that aggregation-caused quenching (ACQ), a common but unfavorable phenomenon in bioimaging, can be turned into an environment-responsive tool to achieve more accurate bioimaging of drug nanocarriers (Qi et al., 2019). The ACQ fluorophores share a BODIPY or aza-BODIPY parent structure with high quantum yield, while they are superior stable and pH insensitive (Hu et al., 2016, 2015; Xie et al., 2018). They emit strong near infrared (NIR) fluorescence in molecular state, but are completely quenched upon contacting with water. This is because the lipophilic probe molecules self-aggregate through intermolecular π-π stacking in aqueous solution, leading to energy dissipation from the excited state via non-photon emitting routes. The ACQ fluorophore is “active” when being molecularly dispersed in a carrier, but “turned off” when being released to the aqueous surroundings. Since water is ubiquitous in the body, the ACQ property enables bioimaging of intact nanocarriers. Similarly, the ACQ probes can be used to identify nanosuspensions by being physically embedded in the drug particles to form the so called hybrid nansuspensions (HNSs) (Liu et al., 2018; Lu et al., 2019; Shen et al., 2018; Xie et al., 2018). In this study, CUR was adopted as a model drug due to its poor dermal permeability and the natural fluorescent property (Beloqui et al., 2014; Wang et al., 2018). In addition, dermal application of CUR has long been of high interest for anti-oxidation, anti-inflammation and photoprotection (Vidlářová et al., 2016). An ACQ probe was physically embedded in the CUR nanosuspensions (CUR-HNSs) for bioimaging. To investigate the size effects on dermal penetration, CUR-HNS of 140 nm (CUR-HNS-140), 400 nm (CUR-HNS-400) and 730 nm (CUR-HNS-730) were prepared, respectively. Since skin is a tissue to control water loss, the epidermis contains 75% of water (Kumar et al., 2012). The skin hydration may be further improved by the occlusive effects from the applied preparations (Ngan et al., 2015). It is rationale to track the transdermal transport of the intact CUR-HNSs using the emission of the ACQ dyes (i.e., the particle signal), because the released dyes are completely quenched in the hydrophilic environment of the skin (Su et al., 2017). Co-localization between the particle and CUR signals provides novel strategy to outline the translocation profiles of intact HNSs as well as the cargoes.

2.1. Materials CUR was purchased from TCI (Tokyo, Japan), while 4′,6-diamidin2-phenylindole (DAPI) from Yeasen Bio-tech Co., Ltd (Shanghai, China). HPMC E5 was a gift from Shanhe Medical Materials Co., Ltd (Anhui, China). The ACQ probe was synthesized in our lab. For detailed structure and fluorescent data of the probe, please refer to (Hu et al., 2015). Deionized water was produced by Milli-Q® (Bedford, MA, USA). All other chemicals and reagents were of analytical grade. Male Sprague-Dawley rats, weighing 180 ± 20 g, were provided by Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and raised in the Laboratory Animal Holding Building of School of Pharmacy, Fudan University. The feeding environment is kept at 24 ± 1 °C and 55 ± 5% relative humidity with natural circadian rhythm of light. Animals were given free diet before experiment. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee at School of Pharmacy, Fudan University (Protocol number: 2019-03-YJ-LY-01). Rules outlined in the Declaration of Helsinki for all human and animal experimental investigations are complied with. 2.2. Preparation of hybrid nanosuspensions The preparation technique and formulation was screened to obtain nanosuspensions of different sizes, which is shown in Supplementary Materials (Figs. S1 and S2). The established fabrication process is as follows: A solvent-antisolvent precipitation method was adopted to prepare CUR-HNS-140 (Sadeghi et al., 2016; Yadav and Kumar, 2014). In brief, 12 mg CUR and 9.6 μg ACQ probe were dissolved in 0.4 mL acetone, which were rapidly mixed with 4.6 mL 0.1% (w/v) HPMC E5 aqueous solution under magnetic stirring at 500 rpm and stirred for 30 s. Nanoparticles were instantly precipitated upon mixing and a uniform yellow suspension was formed. Following evaporation of the organic solvent with a rotary evaporator (R3, Buchi Labortechnik AG, Basel, Switzerland), the suspensions were diluted with 0.1% HPMC E5 solution to a CUR concentration of 2.6 mg/mL. An evaporative precipitation method was developed to prepare CUR-HNS-400 and CUR-HNS-730. For CUR-HNS-400, 13 mg CUR and 10.4 μg ACQ probe were dissolved in 1 mL acetone, which was then mixed with 1.0 mL 0.5% (w/v) HPMC E5 solution to form a transparent solution. CUR nanosuspensions were obtained by evaporating the acetone using the rotary evaporator. The suspensions were diluted with 4 folds of water to get a CUR concentration of 2.6 mg/mL, where the concentration of the ACQ probe and the HPMC in the suspension is equal to that in CUR-HNS-140 suspensions, respectively. The same procedure was adopted to prepare CUR-HNS-730 except that 26 mg CUR, 20.8 μg ACQ probe and 1.0% (w/v) HPMC E5 solution were used. Following the evaporation of acetone, the obtained suspensions were diluted with 9 folds of water to get the equal CUR, ACQ probe and HPMC concentration to that in CUR-HNS-140 and CUR-HNS-400, respectively. 2.3. Characterization Nano® Zetasizer (Malvern Instruments, Malvern, UK) was utilized to measure the size, polydispersity index (PDI) and zeta potential of the CUR-HNSs, following diluting the sample with water by 40 folds. Measurements were performed at 25 °C in triplicate. An S-2460N SEM (Hitachi, Tokyo, Japan) was used to observe the morphologies of the CUR crystals and CUR-HNSs. The CUR-HNSs were diluted with water by 40 folds and then filtered through a 50 nm membrane (Whatman®, GE Healthcare Life Sciences, Pittsburgh, PA, USA). The filtered samples were successively dried in air, transferred to 2

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

microliters of CUR-HNSs or the quenched solution of ACQ probe were added to the donor cell. To prevent loss, the opening of the cell was sealed with parafilm and aluminum foil. In this way, the fluid was restricted to the skin area surrounded by the cell. CUR propylene glycol solution (2.6 mg/mL) and CUR saturated solution in 0.1% HPMC were set as controls. For preparation of the saturated solution, excessive CUR was dispersed in 0.1% (w/v) HPMC solution and stirred for 48 h; the supernatant was collected after centrifugation at 12,000 rpm for 10 min, which was measured to be 92.4 ± 1.80 ng/mL. At 1, 4, 8, 12 and 24 h, the rats were sacrificed by overdose of urethane, respectively. The treated skin was successively cut off, rinsed with normal saline, dried with cotton swabs, frozen, and stored at −20 °C. The frozen skin was cut into 7 μm slices either vertically or horizontally by a microtome (Leica, Mainz, Germany) (Su et al., 2017). The slices were stained with DAPI and observed under LSM 710 confocal fluorescence microscopy (CLSM) (Leica Microsystems, Buffalo Grove, USA). Fluorescence from DAPI, CUR, and ACQ probe was excited by diode laser 405, Argon 488, and He-Ne 633 channel, respectively. The resolution of the scan was set to 1024 × 1024 pixels.

a sample stub, and coated with a thin gold-palladium layer for 60 s by a sputter coater. The observation was performed under an excitation voltage of 5.0 kV. CUR crystals were set as a control. A 204A/G differential scanning calorimeter (DSC) (NETZSCH, Bühl, Germany) was adopted to study the thermal properties of the CUR crystals, HPMC, physical mixture of CUR and HPMC, and CUR-HNSs. To perform the DSC measurement, CUR-HNSs were lyophilized in advance (Xie et al., 2017, 2018). The samples were frozen at −80 °C for 5 h, and then lyophilized at 0.081 mbar for 48 h using a VaCo5 freeze dryer (Zirbus, Niedersachsen, Germany). The lyophilization process didn’t change the particle size of CUR-HNSs after being redispersed in water (data not shown). Approximately 5 mg of sample was sealed in an aluminum pan and heated at 10 °C/min with an empty pan as reference. 2.4. Ex-vivo transdermal permeation A vertical Franz diffusion cell with a diffusion area of 1.77 cm2 was used to evaluate the ex-vivo transdermal permeation of CUR-HNSs. For comparison, CUR propylene glycol solution (2.6 mg/mL) was set as a control. The solubility of CUR in propylene glycol was measured to be 2.84 ± 0.21 mg/mL. Twelve hours before the experiment, the abdominal hair of the rats was carefully removed by electric razor and hair removal creams (Veet, France). Prior to the experiments, the abdominal skin was excised from the rat body following euthanasia with overdose of urethane. The subcutaneous fats and tissues were carefully removed with tweezers. Then the skin was washed with normal saline (Yu et al., 2018). The pretreated skin was fixed between the donor and the receptor chambers with the SC facing upwards. PEG 400 aqueous solution (20%, v/v) was filled in the receptor chamber and stirred at 100 rpm (Zhang et al., 2019). The solubility of curcumin in 20% PEG 400 aqueous solution is 5.03 ± 0.22 μg/mL (37 °C). The receptor chamber was thermostatically maintained at 37 °C to obtain a skin surface temperature of 32 ± 0.5 °C. After 30 min, the receiving media was removed and the receptor chamber was replenished with fresh 20% PEG 400 solution. Aliquots of CUR-HNSs, equivalent to 0.52 mg CUR, were added to the donor chamber. At predetermined time intervals, 1 mL receiving media were withdrawn from the receptor chambers and equal volume of fresh media were immediately added. Following centrifugation at 10,000 rpm for 10 min, the supernatant of each sample was detected by an Agilent 1100 HPLC system (Agilent, Santa Clara, CA, USA) at 425 nm. Separation was performed on a ZORBAX SB-C18 (5 μm, 4.6 mm × 250 mm) column by a mobile phase consisting of 75% methanol and 25% glacial acetic acid solution (3.6%, v/v). The HPLC spectrum is shown in Supplementary Materials, Fig. S3. In the range of 0.25 to 16 μg/mL, the CUR concentration (C) is linear with its peak area (A) with a typical calibration curve of C = 0.0055A + 0.1195, R2 = 0.9997. The detection limit is 0.03 μg/mL. Accuracy of the measurement is 100.05 ± 4.87%. Intra- and inter-day precision were all below 2%. The cumulative permeation amount of CUR (Qn) was calculated as follows (Yu et al., 2018). n−1

Qn = VCn + V0 ∑

i=1

2.6. Statistical analysis One-way analysis of variance (ANOVA) was used to analysis the significance of differences between groups via SPSS statistical software (version 11.0, SPSS, Inc., Chicago, IL, USA). Values of p < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Characterization of CUR-HNSs According to the Zetasizer Nano®, the particle sizes of CUR-HNS140, CUR-HNS-400 and CUR-HNS-730 are 147.1 ± 1.305 nm, 407.1 ± 4.250 nm, and 734.3 ± 19.88 nm, while the PDIs are 0.080 ± 0.014, 0.141 ± 0.031, and 0.053 ± 0.039, respectively (Fig. 1a). The zeta potentials are comparable among the CUR-HNSs, being −28.9 ± 0.252 mV for CUR-HNS-140, −23.9 ± 0.06 mV for CUR-HNS-400, and −23.5 ± 0.78 mV for CUR-HNS-730, respectively. CUR presents rod-like crystals, while all CUR-HNSs are spherical particles with smooth appearance (Fig. 1b). The sizes of the CUR-HNSs are uniform and consistent with the measured results. The morphological transformation may be due to the good glass forming ability of CUR, which leads to lose of crystallinity in antisolvent precipitation (Aditya et al., 2015; Yadav and Kumar, 2014). Materials with strong interactions, e.g., the stabilizers, could further curb the forming of crystals. Therefore, CUR nanosuspension prepared from antisolvent precipitation are in amorphous state and present spherical morphology (Aditya et al., 2015; Lim et al., 2018; Yadav and Kumar, 2014). Similar results are found in materials with good glass forming capability such as cyclosporine A and amphotericin B (Xie et al., 2018; Zhou et al., 2018). The DSC thermograms imply the amorphous state of the CUR-HNSs (Fig. 2). CUR crystal displays a single endothermic peak at 183 °C, corresponding to its melting point. HPMC doesn’t present typical endothermic peaks due to the glassy state. The DSC thermograph of the physical mixture is similar to a superposition of the CUR crystals and HPMC. However, the endothermic peaks disappear in all CUR-HNSs, implying the amorphous state.

Ci

where Cn are the CUR concentrations detected at each intervals, while Ci are the CUR concentration in the ith sampling solution. V and V0 are the volume of the receptor chamber and the sampling solution, respectively. Measurements were performed in triplicate.

3.2. Ex-vivo transdermal permeation 2.5. In vivo transdermal penetration In this part, CUR propylene glycol solution (2.6 mg/mL) was purposely set as a positive control. Provided that molecular diffusion is the main mechanism for transdermal transport from nanosuspensions, the nanosuspensions should not surmount the propylene glycol solution in transdermal permeation, because CUR is molecularly dispersed in the propylene glycol solution while aggregated in amorphous state in the

Similar to the ex-vivo transdermal permeation, the abdominal hair of the rat was removed 12 h prior to the experiment. Following anesthesia by i.p. injection of pentobarbital sodium solution (2.5%, w/v) at a dose of 40 mg/kg, the rats were fixed with fixators. The donor cell of the Franz diffusion cell was adhered on the skin surface. Two hundreds 3

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

Fig. 3. Cumulative transdermal permeation of CUR through the excised rat abdominal skin from CUR-HNSs (n = 3). Table 1 Transdermal permeation parameters of CUR-HNSs. Preparation

Permeation equation

Jss (μg·cm−2·h−1)

R2

CUR-HNS-140 CUR-HNS-400 CUR-HNS-730

Qn/S = 0.3595 t + 1.2020 Qn/S = 0.4310 t + 0.6901 Qn/S = 0.3038 t + 1.0524

0.3595 0.4310 0.3038

0.9749 0.9774 0.9748

from propylene glycol solution were all lower than the linear range of the calibration. This result demonstrates the poor permeability of CUR molecules. Conversely, starting from 2 h, the permeated amounts of CUR from nanosuspensions could be quantified, indicating superior exvivo transdermal permeation to the CUR propylene glycol solution (Fig. 3). But even then, the cumulative amount of permeated CUR is yet less than 2% of the applied dose at 12 h, irrespective of the size of the nanosuspensions. Similar results were found in CUR loaded microemulsions and nano-emulgel, which has been ascribed to the poor permeability of CUR molecules (Jeengar et al., 2016; Sintov, 2015). Table 1 shows the steady state flux (Jss) that is calculated from the linear part of the cumulative permeation per area versus time plot. CUR-HNS-400 presents the highest Jss value followed by CUR-HNS-140 and CUR-HNS-730 in sequence. However, since the permeated amount of CUR is limited, the overall transdermal transport of CUR is comparable among the nanosuspensions. Nonetheless, the results do show an enhanced transdermal transport of CUR from the nanosuspensions than from the propylene glycol solution. Then what are the exact mechanisms?

Fig. 1. Size distribution of CUR-HNSs (a) and the morphologies (b) of CUR crystals (b-1), CUR-HNS-140 (b-2), CUR-HNS-400 (b-3), and CUR-HNS-730 (b4).

3.3. In vivo transdermal penetration To study the mechanisms for the enhanced transdermal effects from CUR-HNSs, co-localization of CUR and the ACQ probe was performed via CLSM observation on the skin treated by the preparations. DAPI (blue signal) was used to stain dermal cells. The CLSM images of rat skin treated with ACQ quenched solution is shown in Supplementary Materials, Fig. S4. Absence of fluorescence indicates no rekindling of the quenched probes due to the disaggregation by dissolving in the lipids of SC. The CUR-HNSs consist of CUR amorphous aggregates and a saturated solution of CUR molecules in 0.1% (v/v) HPMC. Therefore, CUR propylene glycol solution (2.6 mg/mL) and CUR saturated solution in 0.1% (v/v) HPMC were set as controls. Comparison between the saturated solution and CUR-HNSs may distinguish the role of the CUR molecules and the aggregates. Almost no CUR signals are observed in the skin treated by the saturated solution (Supplementary Materials,

Fig. 2. DSC thermograms of CUR-HNS-730 (a), CUR-HNS-400 (b), CUR-HNS140 (c), physical mixtures (d), CUR crystals (e), and HPMC (f).

nanosuspensions. In addition, propylene glycol is a frequently-used transdermal enhancer by enhancing drug partitioning in the skin and creating channels for diffusion (Lopes et al., 2015; Newton, 2013), while HPMC used in the nanosuspensions as a stabilizer is lack of penetrating enhancing effects. However, the permeated amounts of CUR 4

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

demonstrated that fluorescent polystyrene nanoparticles could only infiltrate into 2–3 μm depth of the SC (the outermost, 15–20 μm skin layer), which is independent of contact time (up to 16 h) and of nanoparticle size (20–200 nm) (Campbell et al., 2012); multiphoton microscopy showed no penetration of fluorescently-labelled PLGA nanoparticles (~300 nm) across the SC (Stracke et al., 2006); upconversion nanoparticles ranging from 20 to 24 nm were stopped at SC of human skin (Khabir et al., 2019). The limitation for the ingress of nanoparticles in the SC and the subsequent progression toward the viable epidermis are derived from the nanoporosity in the SC. The staggered corneocyte alignment combined with the intercorneocyte lipidic matrix provides 5–7 nm lipidic intercellular route and maximum 36 nm aqueous pores (Baroli, 2010). Therefore, only nanoparticles smaller than the size limitation may effectively penetrate the SC, such as hydrophilic poly (amidoamine) dendrimers of generation 2 (Yang et al., 2012). Unlike the CUR-HNSs, a gradient CUR signal from viable epidermis to dermis can be seen in the CUR channel of the CLSM photograph (Fig. 5). The reason is attributed to the dissolving of nanosuspensions in the SC and the subsequent diffusion of CUR molecules. The absence of ACQ signals in area below the SC layer supports the diffusion of CUR molecules instead of the intact CUR nanoparticles (the ACQ channel in Fig. 5). Compared with the SC penetration, it is easier for CUR-HNSs to fill in hair follicles (Fig. 5, Figs. S6–S8 in Supplementary Materials, indicated by red arrows), due to the “huge” follicular openings of 10–210 μm in diameter (Baroli, 2010). Similar results have been found in poly(lactic-co-glycolic acid) nanoparticles (100–1000 nm) (Patzelt et al., 2011), polystyrene nanoparticles (40–1500 nm) (Vogt et al., 2006), silica nanoparticles (42–200 nm) (Rancan et al., 2012), nanoemulsions (80–500 nm) (Su et al., 2017), and even microspheres around 1.5 μm (Toll et al., 2004), although the optimal size for hair follicle penetration varies with the nanocarrier type. These findings are correcting the bias against the transfollicular route. The significance of the transfollicular route has been doubted for a long time due to the low density (0.1% of skin surface) and the hindrance from the outward excretion of sweat and sebum. However, the infundibula of the follicles expand the surface area of permeation, while the hair movement provides forces to drive nanoparticles into the follicles (Chen et al., 2018). Similarly, nanosuspensions may have more advantages of delivery efficiency than the abovementioned nanocarriers due to the high drug loading.

Fig. 4. CLSM images of rat skin treated with CUR propylene glycol solution (2.6 mg/mL). Scale bar is 50 μm; the arrow indicates the surface of the skin.

Fig. S5), indicating that transdermal transport from CUR molecules in the nanosupensions is negligible. This is due to the extremely low concentration and poor permeability of CUR molecules. Fig. 4 shows the CLSM images of rat skin treated with CUR propylene glycol solution. Although faint CUR signal is seen in the skin, it is stronger than that in the skin treated by the saturated solution due to the higher CUR concentration and the permeating enhancement of propylene glycol. The bioimaging result also confirms the poor permeability of the CUR molecules. The CLSM images of vertical section of rat skin treated with CURHNSs are shown in Supplementary Materials, Figs. S6–S8. For clarity and to save space, only the skins treated for 4 h are shown in Fig. 5. The red signal represents the intact CUR nanoparticles, while the green represents CUR molecules. The red fluorescence only appears in the SC (indicated by white arrows) and the hair follicles (indicated by red arrows) without obvious differences among CUR-HNSs. It should be noted that DAPI only stains viable epidermis and dermis instead of SC, which consists of dead cells without nucleus. Based on the merged figures, the red fluorescence appears above the outermost edge of the DAPI stained tissue, where the SC layer locates. We infer that a portion of CUR-HNSs can penetrate into the SC instead of entering into the viable epidermis. These results are in agreement with recent literature on transdermal penetration of nanoparticles. CLSM imaging

Fig. 5. CLSM images of vertical section of rat skin treated with CUR-HNSs at 4 h post administration (white arrows, stratum corneum; red arrows, hair follicles). Dashed lines in the insert outline the translocation profiles of CUR-HNSs. Scale bar is 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 5

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

The CLSM images of horizontal section of skin samples 4 h post administration confirm the transfollicular transport of CUR-HNSs (Fig. 6). Bright spots, instead of planes, present in the slides, being attributed to the accumulation of CUR-HNSs in hair follicles. Unlike other types of nanoparticles, size dependent hair follicle penetration is not seen among CUR-HNSs. Compared with the huge follicular openings (10 to 210 μm), the gaps of the sizes among CUR-HNSs are negligible. The ACQ signals are lightened along the depth of the hair follicles till 154–175 μm (Supplementary Materials, Figs. S9–S11), indicating dissolving of CUR-HNSs in the hair follicles. Conversely, the CUR signals can be seen in the depth of 196 μm and in a constant intensity due to the environmental insensitivity. Most importantly, CUR signals are observed in area around the hair follicles, indicating the diffusion of CUR molecules to peri-follicular tissues. Due to the absence of a SC barrier, the lower follicular orifice provides a permeable sites for CUR molecules (Mittal et al., 2013; Su et al., 2017). However, the hair follicle accumulation is minor for CUR propylene glycol solution (Fig. 4). It is thus rational to infer that the accumulation of CUR-HNSs in the hair follicles and the subsequent diffusion of CUR molecules account for the enhanced transdermal delivery from nanosuspensions. To sum up, combination of the results from vertical and horizontal section outlines the translocation profiles of intact CUR-HNSs, being drawn by dashed lines in Fig. 5 insert. The intact CUR-HNSs can not only infiltrate into the SC layer, but also accumulate in the hair follicles. But they cannot enter into the viable epidermis and dermis. CUR molecules are released due to dissolving of CUR-HNSs that reside in the SC and the hair follicles. Unlike the intact CUR-HNSs, the CUR molecules diffuse passively into the skin. However, it is unable to discriminate the dominating route for dermal diffusion at present.

4. Conclusions Different sizes of CUR-HNSs have been prepared by either antisolvent or evaporative precipitation technique in this study to investigate the transdermal mechanisms of nanosuspensions. CUR-HNSs show enhanced transdermal delivery of CUR than the solution counterpart. Although CUR-HNS-400 presents the highest Jss, the overall transdermal transport of CUR is comparable among the nanosuspensions due to the limited permeated amount, i.e., less than 2% of the applied dose at 12 h. ACQ properties can be used to identify the intact CUR nanoparticles. The co-localization of the particle and CUR signals outlines the translocation profiles of intact HNSs, as well as the cargoes. CUR-HNSs can either infiltrate into the SC layer or accumulate in the hair follicles, where the nanosuspenions dissolve and allow CUR molecules diffuse into the skin. For this reason, the SC barrier hindering permeation of CUR molecules is surmounted. In conclusion, nanosuspensions are potential in transdermal delivery of drug substances that are both poorly permeable and poorly soluble in water, which, however, needs to be further verified in human skins.

CRediT authorship contribution statement Tingting Shi: Investigation. Yongjiu Lv: Visualization. Weizi Huang: Investigation. Zhezheng Fang: Investigation. Jianping Qi: Funding acquisition. Zhongjian Chen: Resources. Weili Zhao: Resources. Wei Wu: Conceptualization, Funding acquisition. Yi Lu: Conceptualization, Funding acquisition, Writing - original draft. Fig. 6. CLSM images of horizontal section of rat skin treated with CUR-HNS140 (a), CUR-HNS-400 (b), CUR-HNS-730 (c) for 4 h. For a whole set of images of all depths, please refer to Fig. S9–S11 in Supplementary Materials. Scale bar is 50 μm.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

Acknowledgements

Lu, Y., Qi, J., Dong, X., Zhao, W., Wu, W., 2017. The in vivo fate of nanocrystals. Drug Discov. Today 22, 744–750. Mak, W.C., Richter, H., Patzelt, A., Sterry, W., Lai, K.K., Renneberg, R., Lademann, J., 2011. Drug delivery into the skin by degradable particles. Eur. J. Pharm. Biopharm. 79, 23–27. Malamatari, M., Taylor, K.M.G., Malamataris, S., Douroumis, D., Kachrimanis, K., 2018. Pharmaceutical nanocrystals: production by wet milling and applications. Drug Discov. Today 23, 534–547. Mittal, A., Raber, A.S., Lehr, C.M., Hansen, S., 2013. Particle based vaccine formulations for transcutaneous immunization. Hum. Vaccin. Immunother. 9, 1950–1955. Mohammad, I.S., Hu, H., Yin, L., He, W., 2019a. Drug nanocrystals: Fabrication methods and promising therapeutic applications. Int. J. Pharm. 562, 187–202. Mohammad, I.S., Teng, C., Chaurasiya, B., Yin, L., Wu, C., He, W., 2019b. Drug-delivering-drug approach-based codelivery of paclitaxel and disulfiram for treating multidrug-resistant cancer. Int. J. Pharm. 557, 304–313. Newton, S.J., 2013. Chemical penetration enhancers. Int. J. Pharm. Compd. 17, 370–374. Ngan, C.L., Basri, M., Tripathy, M., Abedi Karjiban, R., Abdul-Malek, E., 2015. Skin intervention of fullerene-integrated nanoemulsion in structural and collagen regeneration against skin aging. Eur. J. Pharm. Sci. 70, 22–28. Patzelt, A., Mak, W.C., Jung, S., Knorr, F., Meinke, M.C., Richter, H., Rühl, E., Cheung, K.Y., Tran, N.B.N.N., Lademann, J., 2017. Do nanoparticles have a future in dermal drug delivery? J. Control. Release 246, 174–182. Patzelt, A., Richter, H., Knorr, F., Schafer, U., Lehr, C.M., Dahne, L., Sterry, W., Lademann, J., 2011. Selective follicular targeting by modification of the particle sizes. J. Control. Release 150, 45–48. Qi, J., Hu, X., Dong, X., Lu, Y., Lu, H., Zhao, W., Wu, W., 2019. Towards more accurate bioimaging of drug nanocarriers: turning aggregation-caused quenching into a useful tool. Adv. Drug Deliv. Rev. 143, 206–225. Rancan, F., Gao, Q., Graf, C., Troppens, S., Hadam, S., Hackbarth, S., Kembuan, C., Blume-Peytavi, U., Ruhl, E., Lademann, J., Vogt, A., 2012. Skin penetration and cellular uptake of amorphous silica nanoparticles with variable size, surface functionalization, and colloidal stability. ACS Nano 6, 6829–6842. Sadeghi, F., Ashofteh, M., Homayouni, A., Abbaspour, M., Nokhodchi, A., Garekani, H.A., 2016. Antisolvent precipitation technique: A very promising approach to crystallize curcumin in presence of polyvinyl pyrrolidon for solubility and dissolution enhancement. Colloid. Surf. B 147, 258–264. Shen, C., Yang, Y., Shen, B., Xie, Y., Qi, J., Dong, X., Zhao, W., Zhu, W., Wu, W., Yuan, H., Lu, Y., 2018. Self-discriminating fluorescent hybrid nanocrystals: efficient and accurate tracking of translocation via oral delivery. Nanoscale 10, 436–450. Sinico, C., Pireddu, R., Pini, E., Valenti, D., Caddeo, C., Fadda, A.M., Lai, F., 2017. Enhancing topical delivery of resveratrol through a nanosizing approach. Planta Med. 83, 476–481. Sintov, A.C., 2015. Transdermal delivery of curcumin via microemulsion. Int. J. Pharm. 481, 97–103. Stracke, F., Weiss, B., Lehr, C.M., Konig, K., Schaefer, U.F., Schneider, M., 2006. Multiphoton microscopy for the investigation of dermal penetration of nanoparticleborne drugs. J, Invest. Dermatol. 126, 2224–2233. Su, R., Fan, W., Yu, Q., Dong, X., Qi, J., Zhu, Q., Zhao, W., Wu, W., Chen, Z., Li, Y., Lu, Y., 2017. Size-dependent penetration of nanoemulsions into epidermis and hair follicles: implications for transdermal delivery and immunization. Oncotarget 8, 38214–38226. Teichmann, A., Otberg, N., Jacobi, U., Sterry, W., Lademann, J., 2006. Follicular penetration: development of a method to block the follicles selectively against the penetration of topically applied substances. Skin Pharmacol. Physiol. 19, 216–223. Toll, R., Jacobi, U., Richter, H., Lademann, J., Schaefer, H., Blume-Peytavi, U., 2004. Penetration profile of microspheres in follicular targeting of terminal hair follicles. J. Invest. Dermatol. 123, 168–176. Tsai, M.J., Fu, Y.S., Lin, Y.H., Huang, Y.B., Wu, P.C., 2014. The effect of nanoemulsion as a carrier of hydrophilic compound for transdermal delivery. PLoS ONE 9, e102850. Vidlářová, L., Romero, G.B., Hanuš, J., Štěpánek, F., Müller, R.H., 2016. Nanocrystals for dermal penetration enhancement – Effect of concentration and underlying mechanisms using curcumin as model. Eur. J. Pharm. Biopharm. 104, 216–225. Vogt, A., Combadiere, B., Hadam, S., Stieler, K.M., Lademann, J., Schaefer, H., Autran, B., Sterry, W., Blume-Peytavi, U., 2006. 40 nm, but not 750 or 1,500 nm, nanoparticles enter epidermal CD1a+ cells after transcutaneous application on human skin. J. Invest. Dermatol. 126, 1316–1322. Wang, T., Qi, J., Ding, N., Dong, X., Zhao, W., Lu, Y., Wang, C., Wu, W., 2018. Tracking translocation of self-discriminating curcumin hybrid nanocrystals following intravenous delivery. Int. J. Pharm. 546, 10–19. Wang, Y., Zhang, Y., Wang, J., Liang, X.J., 2019. Aggregation-induced emission (AIE) fluorophores as imaging tools to trace the biological fate of nano-based drug delivery systems. Adv. Drug Deliv. Rev. 143, 161–176. Wu, W., Li, T., 2019. Unraveling the in vivo fate and cellular pharmacokinetics of drug nanocarriers. Adv. Drug Deliv. Rev. 143, 1–2. Xie, Y., Chen, Z., Su, R., Li, Y., Qi, J., Wu, W., Lu, Y., 2017. Preparation and optimization of amorphous ursodeoxycholic acid nanosuspensions by nanoprecipitation based on acid-base neutralization for enhanced dissolution. Curr. Drug Delivery 14, 483–491. Xie, Y., Shi, B., Xia, F., Qi, J., Dong, X., Zhao, W., Li, T., Wu, W., Lu, Y., 2018. Epithelia transmembrane transport of orally administered ultrafine drug particles evidenced by environment sensitive fluorophores in cellular and animal studies. J. Control. Release 270, 65–75. Xin, X., Pei, X., Yang, X., Lv, Y., Zhang, L., He, W., Yin, L., 2017. Rod-shaped active drug particles enable efficient and safe gene delivery. Adv. Sci. 4, 1700324. Yadav, D., Kumar, N., 2014. Nanonization of curcumin by antisolvent precipitation: process development, characterization, freeze drying and stability performance. Int. J. Pharm. 477, 564–577.

This work is financially supported by National Natural Science Foundation of China (81973247, 81872815, 81872826 and 81690263) and Science and Technology Commission of Shanghai Municipality (19430741400, 19410761200, 19XD1400300). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2020.119737. References Aditya, N.P., Yang, H., Kim, S., Ko, S., 2015. Fabrication of amorphous curcumin nanosuspensions using β-lactoglobulin to enhance solubility, stability, and bioavailability. Colloid. Surf. B 127, 114–121. Alexander, A., Dwivedi, S., Ajazuddin, Giri, T.K., Saraf, S., Saraf, S., Tripathi, D.K., 2012. Approaches for breaking the barriers of drug permeation through transdermal drug delivery. J. Control. Release 164, 26–40. Baroli, B., 2010. Penetration of nanoparticles and nanomaterials in the skin: fiction or reality? J. Pharm. Sci. 99, 21–50. Beloqui, A., Coco, R., Memvanga, P.B., Ucakar, B., des Rieux, A., Preat, V., 2014. pHsensitive nanoparticles for colonic delivery of curcumin in inflammatory bowel disease. Int. J. Pharm. 473, 203–212. Campbell, C.S., Contreras-Rojas, L.R., Delgado-Charro, M.B., Guy, R.H., 2012. Objective assessment of nanoparticle disposition in mammalian skin after topical exposure. J. Control. Release 162, 201–207. Chen, T., He, B., Tao, J., He, Y., Deng, H., Wang, X., Zheng, Y., 2019. Application of Forster Resonance Energy Transfer (FRET) technique to elucidate intracellular and in vivo biofate of nanomedicines. Adv. Drug Deliv. Rev. 143, 177–205. Chen, Z., Lv, Y., Qi, J., Zhu, Q., Lu, Y., Wu, W., 2018. Overcoming or circumventing the stratum corneum barrier for efficient transcutaneous immunization. Drug Discov. Today 23, 181–186. Chen, Z., Wu, W., Lu, Y., 2020. What is the future for nanocrystal-based drug-delivery systems? Ther. Deliv. 11, 225–229. Corrias, F., Schlich, M., Sinico, C., Pireddu, R., Valenti, D., Fadda, A.M., Marceddu, S., Lai, F., 2017. Nile red nanosuspensions as investigative model to study the follicular targeting of drug nanocrystals. Int. J. Pharm. 524, 1–8. Couto, A., Fernandes, R., Cordeiro, M.N., Reis, S.S., Ribeiro, R.T., Pessoa, A.M., 2014. Dermic diffusion and stratum corneum: a state of the art review of mathematical models. J. Control. Release 177, 74–83. Giannos, S.A., 2015. Identifying present challenges to reliable future transdermal drug delivery products. Ther. Deliv. 6, 1033–1041. Hassan, A.O., Elshafeey, A.H., 2010. Nanosized particulate systems for dermal and transdermal delivery. J. Biomed. Nanotechnol. 6, 621–633. Hu, X., Fan, W., Yu, Z., Lu, Y., Qi, J., Zhang, J., Dong, X., Zhao, W., Wu, W., 2016. Evidence does not support absorption of intact solid lipid nanoparticles via oral delivery. Nanoscale 8, 7024–7035. Hu, X., Zhang, J., Yu, Z., Xie, Y., He, H., Qi, J., Dong, X., Lu, Y., Zhao, W., Wu, W., 2015. Environment-responsive aza-BODIPY dyes quenching in water as potential probes to visualize the in vivo fate of lipid-based nanocarriers. Nanomedicine 11, 1939–1948. Jeengar, M.K., Rompicharla, S.V., Shrivastava, S., Chella, N., Shastri, N.R., Naidu, V.G., Sistla, R., 2016. Emu oil based nano-emulgel for topical delivery of curcumin. Int. J. Pharm. 506, 222–236. Khabir, Z., Guller, A.E., Rozova, V.S., Liang, L., Lai, Y.J., Goldys, E.M., Hu, H., Vickery, K., Zvyagin, A.V., 2019. Tracing upconversion nanoparticle penetration in human skin. Colloid. Surf. B 184, 110480. Kumar, A., Pathak, K., Bali, V., 2012. Ultra-adaptable nanovesicular systems: a carrier for systemic delivery of therapeutic agents. Drug Discov. Today 17, 1233–1241. Li, C., Wang, J., Wang, Y., Gao, H., Wei, G., Huang, Y., Yu, H., Gan, Y., Wang, Y., Mei, L., Chen, H., Hu, H., Zhang, Z., Jin, Y., 2019. Recent progress in drug delivery. Acta Pharm. Sin. B 9, 1145–1162. Li, Y., Wang, D., Lu, S., Zeng, L., Wang, Y., Song, W., Liu, J., 2018. Pramipexole nanocrystals for transdermal permeation: Characterization and its enhancement micromechanism. Eur. J. Pharm. Sci. 124, 80–88. Lim, L.M., Tran, T.T., Long Wong, J.J., Wang, D., Cheow, W.S., Hadinoto, K., 2018. Amorphous ternary nanoparticle complex of curcumin-chitosan-hypromellose exhibiting built-in solubility enhancement and physical stability of curcumin. Colloid. Surf. B 167, 483–491. Liu, D.L., Wan, B., Qi, J.P., Dong, X.C., Zhao, W.L., Wu, W., Dai, Y.K., Lu, Y., Chen, Z.J., 2018. Permeation into but not across the cornea: Bioimaging of intact nanoemulsions and nanosuspensions using aggregation-caused quenching probes. Chin. Chem. Lett. 29, 1834–1838. Lopes, L.B., Garcia, M.T., Bentley, M.V., 2015. Chemical penetration enhancers. Ther. Deliv. 6, 1053–1061. Lu, Y., Chen, Y., Gemeinhart, R.A., Wu, W., Li, T., 2015. Developing nanocrystals for cancer treatment. Nanomedicine 10, 2537–2552. Lu, Y., Li, Y., Wu, W., 2016. Injected nanocrystals for targeted drug delivery. Acta Pharm. Sin. B 6, 106–113. Lu, Y., Lv, Y., Li, T., 2019. Hybrid drug nanocrystals. Adv. Drug Deliv. Rev. 143, 115–133.

7

International Journal of Pharmaceutics 588 (2020) 119737

T. Shi, et al.

topical anti-psoriatic efficacy of curcumin-loaded hyaluronan-modified ethosomes: a new strategy for clustering drug in inflammatory skin. Theranostics 9, 48–64. Zhou, Y., Fang, Q., Niu, B., Wu, B., Zhao, Y., Quan, G., Pan, X., Wu, C., 2018. Comparative studies on amphotericin B nanosuspensions prepared by a high pressure homogenization method and an antisolvent precipitation method. Colloid. Surf. B 172, 372–379.

Yang, Y., Sunoqrot, S., Stowell, C., Ji, J., Lee, C.-W., Kim, J.W., Khan, S.A., Hong, S., 2012. Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration. Biomacromolecules 13, 2154–2162. Yu, Q., Wu, X., Zhu, Q., Wu, W., Chen, Z., Li, Y., Lu, Y., 2018. Enhanced transdermal delivery of meloxicam by nanocrystals: Preparation, in vitro and in vivo evaluation. Asian J. Pharm. Sci. 13, 518–526. Zhang, Y., Xia, Q., Li, Y., He, Z., Li, Z., Guo, T., Wu, Z., Feng, N., 2019. CD44 assists the

8