Materials
LA and RAN (purity >98%, each) were purchased from Shaanxi Chenguang Pharmaceutical Co., Ltd. (Xi’an, China). AS was purchased from Shaanxi Minsheng Medicine Co., Ltd. (Xi’an, China). Caprylocaproyl macrogol-8 glycerides (Labrasol®) and glyceryl palmitostearate (Precirol® ATO 5) were provided by Gattefossé (Montesquieu, France). Polyoxyl castor oil (Cremophor® RH40) was obtained from BASF (Ludwigshafen, Germany). High-glucose Dulbecco’s modified Eagle medium (DMEM-high) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Trypsin (0.25%), 0.02% ethylenediaminetetraacetic acid (EDTA), fetal calf serum (FCS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and phosphate-buffered saline (PBS) were obtained from Shanghai Usen Biotechnology (Shanghai, China). Filipin III, genistein, cytochalasin D, 5-(N,N-dimethyl)-amiloride hydrochloride, monensin sodium, Lyso-Tracker® Red DND-99, and Hoechst33258 stain were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Sodium azide was obtained from Beijing Bei-na Kai-chuang Biotech Co., Ltd (Beijing, China), and chlorpromazine hydrochloride was obtained from Aladdin Industrial Inc. (Shanghai, China). All other chemicals were obtained from Sinopharm Chemical Reagent (Shanghai, China) and were of high-performance liquid chromatography (HPLC) or analytical grade.
Animals and cell lines
Male Sprague–Dawley rats and nude mice weighing 200 ± 20 and 25 ± 5 g, respectively were used in this study, which was conducted with the approval of the Animal Ethical Committee of the Shanghai University of Traditional Chinese Medicine (permit SYXK [Hu] 2009-0069). All the animals were acclimatized at a temperature of 25 ± 2°C and relative humidity of 70 ± 5% for 1 week with food and water provided ad libitum. The HaCaT cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the CCC-ESF cell line was obtained from the Chinese Academy of Medical Sciences (Beijing, China).
HPLC analysis
LA and RAN were analyzed using an Agilent HPLC system (HP 1260, Agilent Technologies, Inc., Santa Clara, USA) with an octadecylsilyl column (Inspire™ C18 (Dikma Technologies, Beijing, China), 250 mm × 4.6 mm, 5 µm). The mobile phase consisted of a solution of methanol and 0.01 mol L−1 monosodium phosphate aqueous solution (80:20, v/v) at a flow rate of 1.0 mL min−1. The column temperature was maintained at 30°C and the detector was set at 252 nm. Quantified samples were filtered through a 0.45-µm filter membrane before automatic injection into the HPLC system.
Preparation of AAS
AAS was extracted using alcohol reflux extraction methods. Briefly, powdered AS was soaked in acidified water (pH 1)/90% ethanol (v/v) at a ratio of 1:10 (w/v), and then extracted by reflux with heating for 2 h in a water bath at 100°C. The extract was collected, and then replaced with fresh solvent. This extraction procedure was repeated twice. After filtration and alkalization (pH > 10) with ammonium hydroxide, the extracts were further treated with chloroform and concentrated by vacuum distillation. To prepare the AAS, the residue was dissolved in acetone and recrystallized with diethyl ether, and the obtained extracts were vacuum-dried (relative vacuum, −0.1 MPa; temperature: 60°C) for 12 h. The main components of AAS, which have superior pharmacological effects were LA and RAN (yield, LA 69.47 and RAN 9.16%, w/w, respectively).
Ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) analysis
The in vivo dialysate samples were analyzed using a Thermo Ultimate 3000 ultra-performance liquid chromatography system coupled to a Thermo TSQ Quantum triple-quadrupole tandem mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with an electrospray ionization source. The Xcalibur 1.2 data analysis system was used. Chromatographic separation was achieved on an octadecylsilyl column [Hypersil GOLD™ C18 (Thermo Finnigan, San Jose, CA, USA), 100 mm × 2.1 mm, 5 µm] maintained at 30°C. The mobile phase consisted of methanol:water:acetic acid (70:30:0.2, v/v/v) with a flow rate of 0.2 mL/min, and the injection volume was 5 µL. The mass spectrometer was operated in the positive mode. The spray voltage was set to 3,500 kV and the capillary temperature was set at 350°C. Nitrogen was used as the sheath (35 psi) and auxiliary gas (10 psi). The precursor-to-production transitions were monitored at m/z 585 → 356 for LA and m/z 601 → 422 for RAN using the selected reaction monitoring mode. Quantified samples were extracted with diethyl ether and dissolved in methanol before automatic injection into the UPLC-MS/MS.
Preparation of AAS-NLCs
The different AAS-NLC formulations were prepared using the hot-melt high-pressure homogenization method [32] with Precirol ATO 5, labrasol, and Cremophor RH40 as the solid lipid, liquid lipid, and surfactant, respectively. Briefly, AAS was dissolved in the solid (Precirol ATO 5) and liquid (labrasol) lipids, which were melted in a water bath at 75°C. The required amount of the aqueous phase (Cremophor RH40 in double-distilled water) was then added to the oleic phase dropwise under high-speed mixing using a disperser (Ultra Turrax T25, IKA, Staufen, Germany). The resulting pre-emulsion was then immediately passed through a high-pressure homogenizer (NS1001 L, GEA, Parma, Italy). To optimize the NLC formulations, a uniform design with a U*7(74) table was used in this study that included the following factors: the amount of lipids (solid and liquid lipids, factor A), the amount of surfactant (factor B), the ratio of solid lipids to liquid lipids (factor C), and the ratio of drugs to lipids (factor D). This scheme produced seven NLC formulations (NLC1–NLC7) that were characterized with regards to particle size, EE, and drug DL. The AAS-SLN preparations used for comparison were prepared using the same method with Precirol ATO5 (9.0%, w/v), Cremophor RH40 (5.5%, w/v), and the same drug content.
Characterization of AAS-NLCs
Particle size and zeta potential (ZP)
A computerized Malvern Autosizer Nano ZS90 inspection system (Malvern Instruments, Malvern, UK) was used to measure the average particle size and zeta potential (ZP) of each preparation via dynamic light scattering. The samples were diluted with distilled water before measurements were taken and each was performed in triplicate.
EE and DL
The free drug was separated using an ultrafiltration method [33]. Centrifugal filter tubes (10 kDa, Pall Corporation, Port Washington, NY, USA) were used to estimate the EE and DL. Nanoparticle suspensions (0.5 mL) were placed in the centrifugal filter tubes, and centrifuged for 15 min at 5,000 rpm. The free drug was separated from the drug entrapped in the nanoparticles and collected at the bottom of the tube through the membrane. The separated and total drug contents were evaluated using a validated HPLC. The total drug content in each preparation was calculated after extraction with acetonitrile in an ultrasonic bath. The EE and DL were calculated using the following equations:
$${\text{EE}}\% \, = \, \left( {W_{total} - W_{free} } \right)/W_{total} \times \, 100$$
(1)
$${\text{DL}}\% \, = \, \left( {W_{total} - W_{free} } \right)/W_{lipids} \times \, 100$$
(2)
where, W
total
, W
free
, and W
lipids
are the total amount of LA and RAN in the preparation, the amount of untrapped LA and RAN, and the amount of lipids used in the formulation, respectively.
Tem
The physical appearance of the AAS-NLCs were examined and with that of the AAS-SLNs using TEM (JEM-1230; Jeol, Ltd., Tokyo, Japan). The samples were prepared for negative staining as follows. The samples were diluted with distilled water and placed on a film-coated copper grid (Zhong Jing Ke Yi Technology Inc., Beijing, China) to dry for about 20 min. Next, a drop of 2% phosphotungstic acid was added to the film and allowed to dry for 10 min, after which the sample was examined under the TEM.
Dsc
DSC was performed using a differential scanning calorimeter (Shimadzu DSC-60; Shimadzu Corporation, Kyoto, Japan). The sample (lyophilized in advance using mannitol 10%, w/v as the cryoprotectant) was heated at the scanning rate of 10°C per min over a temperature range of 30–300°C. An empty aluminum pan was used as a reference for comparison.
Xrd
XRD analysis of the AAS-loaded lipid nanoparticles (lyophilized in advance using mannitol 10%, w/v as the cryoprotectant) was performed using an X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) to assess their crystalline structures. The X-ray diffraction patterns were recorded at 2θ values of 3–50° at a scanning speed of 5° per min using a Cu-Kα radiation source.
In vitro release
The dialysis method was used to assess the in vitro drug release pattern [34]. Dialysis bags (molecular weight cutoff, 14 kDa, Pall Corporation, Port Washington, NY, USA) containing 5 mL of the test formulations were firmly tied and immersed in 100 mL of normal saline containing 20% polyethylene glycol 400 (PEG 400, v/v) at 37 ± 0.5°C and stirred at 120 rpm with a paddle. At each sampling time point, 1 mL of the sample was withdrawn, and then replaced with the same volume of fresh medium. The withdrawn samples were measured using HPLC. All experiments were performed in triplicate.
In vitro cell uptake
Cell culture
HaCaT and CCC-ESF were grown in fresh culture medium (DMEM-high) containing 10% FCS (v/v) and maintained at 37°C under humidified conditions with 5% CO2 (Forma 3111, Thermo Fisher Scientific). For the subculture, cells growing as a monolayer were detached from the culture dish with 0.05% (w/v) trypsin solution. All cell-handling procedures were performed on a super-clean bench (VCM-620; Dabao Instrument, Suzhou, China) and aseptic techniques were employed.
Cytotoxicity
The cytotoxicity of free drugs, AAS-NLCs, AAS-SLNs, and empty vehicles was determined using the MTT assay [35]. The AAS solution was prepared using dimethylsulfoxide solution and culture medium (DMEM-high) while the AAS-NLC preparations, AAS-SLN preparations, and empty vehicle suspension were prepared in culture medium (DMEM-high) over a range of 50–1,000 µg/mL. The working solutions at various concentrations were added to cells cultured in 96-well microplates at 5 × 103 cells/well and incubated for 4 h. Then, the culture fluid was replaced with fresh DMEM (no FBS) containing 500 μg/mL MTT. After a 4-h incubation at 37°C, the culture medium was removed and replaced with 200 μL dimethylsulfoxide. The plate was shaken for 10 min in the dark and the absorbance was determined at 570 nm in a microplate reader (Biotek Synergy HT, Gene Company Limited, Hong Kong, China). All measurements were performed in triplicate and results were calculated as a percentage of the values obtained from the control wells (untreated cells).
Cellular uptake
Cellular uptake was evaluated using flow cytometry with coumarin-6 (C6) dehydrated alcohol solution (1.0 mg/mL) instead of AAS. NLC and the compared SLN formulations containing 10 µg/mL C6 were prepared. Cells were incubated with the C6-labeled formulations in the culture dish for 15, 60, and 120 min, after which the culture medium was removed and the cells were rinsed thrice with 1 mL PBS (pH 7.4). After trypsinization, the cells were carefully collected into 5-mL BD tubes (Falcon 352002; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and centrifuged at 1,500 rpm at 25°C in a refrigerated centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was removed and the cells were resuspended in 0.5 mL of PBS (pH 7.4, containing 1% FCS, v/v) in the BD tubes. A total of 20,000 cells were analyzed using a flow cytometer (FCM, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cells that had not been incubated with any fluorophore served as the blank control cells.
Endocytic pathway
Endocytosis mechanisms were assessed using cellular uptake assays in the presence of chemicals known to inhibit specific endocytosis pathways [36]. Cells were incubated with CPZ, FLP, GNT, CCD, DMA, NaN3, or MS (at 10, 5, 54, 1.52, 2.66, and 2.08 µg/mL, respectively) for 30 min at 37°C. After exposure to these inhibitors, 200 µL of C6-labeled NLCs was added to each well and incubated for 30 min. After the incubation, cells were prepared for flow cytometry as described earlier and the data for at least 20,000 cells were acquired. The cells treated with C6-labeled NLCs alone were considered as the control cells.
Confocal imaging
First, 100 µL of the culture medium (no FCS) in glass-bottom culture dishes (MatTek P35G-0-10-C35 mm, MatTek Corporation, Ashland, USA) was removed and replaced with an equal volume of either the C6-labeled NLC suspension or the C6-labeled SLN suspension. After a 30-min incubation in 5% CO2 atmosphere maintained at 37°C, the culture fluid was removed, and the culture dish was washed thrice with 1 mL pre-cooled PBS (pH 7.4). Fresh DMEM (no FBS) containing 50 nM LysoTracker Red was added to each sample and the cells were incubated for 30 min at 37°C. Hoechst 33342 was added to a final concentration of 10 μg/mL and the cells were cultured for a further 5 min at 37°C. The culture fluid was removed rapidly, 1 mL of aqueous paraformaldehyde solution (4%, w/v) was added to the culture dish to fix the cells for 15 min, the fixative was removed, and then the cells were washed thrice with 1 mL PBS (pH 7.4). The cells were optically scanned within 2 h at different increments through the z-axis with a TCS SP5 confocal imager (Leica Microsystems, Wetzlar, Germany).
In vitro transdermal studies
Rats were anesthetized, euthanized, their abdominal fur was removed with a razor, and then the skin was excised carefully and washed with normal saline. In vitro permeation experiments were conducted using a Franz diffusion cell (Fulansi Electronic Science and Trade Co, Ltd, Tianjin, China) fitted with excised rat skin [37]. Each donor compartment had a diffusion area of 2.0 cm2 and each receptor compartment was filled with 12.5 mL of freshly prepared normal saline containing 20% PEG 400 (v/v) to provide sink conditions [38]. The saline in the receptor compartment was maintained at a temperature of 37 ± 0.5°C and stirred at 500 rpm using a magnetic bar. One mL of each formulation was added to the donor compartment, and it was sealed with parafilm. At predetermined time points, 1 mL of the sample was removed from the receptor compartment, and replaced with an equal volume of receptor fluid equilibrated to 37 ± 0.5°C. The permeation studies were performed in triplicate and the collected samples were analyzed using HPLC.
In vitro fluorescence detection-based NTA in receptor fluid
A Franz diffusion cell fitted with excised rat skin was prepared as described earlier. The receptor compartment was filled with purified water containing 0.02% NaN3 and the donor compartment was filled with 1.0 mL of the C6-labeled NLC suspension, C6-labeled SLN suspension, or 1.0 mL of C6 suspended in distilled water as a blank control sample. The diffusion cell was stirred at 500 rpm while the temperature was maintained at 37 ± 0.5°C. After 12 h, samples were removed from each receptor compartment and measured using a NanoSight NS300 system (Malvern Instruments, Malvern, UK), which was equipped with a scientific sCMOS camera, a 405 nm laser with temperature control, and a 503 nm long-pass filter for fluorescent particle analysis. NanoSight NS300 NTA 3.0 software was used for the data collection and the temperature was maintained at 25°C throughout the experiment.
Effect on SC structure
A nude mouse was anesthetized and immobilized on a mat with the abdomen facing upward, and then the skin in this region was divided into three areas. A flat cylindrical plastic cover with an area of 1 cm2 that served as a drug pool was glued above each skin area using cyanoacrylate adhesive. One mL of NLCs or SLNs was applied to the plastic cover, and the untreated skin area was maintained as a control region. After 8 h, the mouse was euthanized, and the abdominal skin was collected and washed with normal saline. The skin samples were fixed in glutaraldehyde for 24 h, rinsed with PBS (pH 7.4), fixed in 1% osmic acid, dehydrated with ethanol, and then dried using CO2 critical point drying. Finally, the skin was sputtered with platinum and evaluated using a scanning electron microscope (SEM, Quanta FEG250, FEI, Hillsboro, OR, USA).
Microdialysis system
The microdialysis system used was composed of a WZ-50C6 microinfusion pump (Smiths Medical, St. Paul, MN, USA) with a 20-mL plastic syringe and a linear microdialysis probe. The regenerated cellulose (inner diameter, 200 µm; outer diameter, 280 µm; molecular weight cut-off, 13 kDa) was used to prepare Spectra/Por® microdialysis hollow fibers (Spectrum Laboratories, Inc., Houston, TX, USA). The linear microdialysis probes were made by gluing the fibers to the quartz capillary tubing (Welch Materials, Inc., Shanghai, China) using cyanoacrylate adhesive (Mxbon® Super Glue, Cartell Chemical Co., Ltd., Chia-Yi Hsien, Taiwan). The inlet tubes of the probes were connected to the microinjection pump using polyethylene tubing [39]. In all experiments, the length of the membrane accessible to dialysis was 20 mm, and the perfusate flow rate was 0.2 mL/h. Cannulas were used as insertion guides and vials were used to collect the dialysate samples.
Standard (STD) solutions of LA and RAN were prepared by dissolving pure LA and RAN in normal saline containing 20% PEG400 (v/v). The concentrations of the STD solutions (STD1–STD6) were 1, 5, 10, 25, 50, and 100 ng/mL, respectively.
In vivo recovery validation in vivo correction
Before the microdialysis studies, in vitro recovery was estimated to ensure that the probes would provide reproducible and efficient sampling. The dialysis membrane portion of the linear probe was completely submerged in a standard solution of LA or RAN (STD, prepared by dissolving pure LA or RAN in 20% PEG 400 in normal saline, v/v) at 37°C in a 50-mL beaker. The probe was perfused with 20% PEG 400 in normal saline (v/v) at a flow rate of 0.2 mL/h. After an equilibration period of 30 min, the dialysate was collected in a small vial for 30 min and dialysate samples were analyzed using UPLC-MS/MS. Relative recovery (RR
d
) was calculated as the slope of the linear regression of the drug concentration in the dialysate (C
d
) as a function of the drug concentration in the medium (C
m
):
$${\text{RR}}_{d} = C_{d} /C_{m} \times { 1}00$$
(3)
For the retrodialysis studies, the probe was perfused with STD solutions (C
p
) and the medium was replaced with 20% PEG 400 in normal saline (v/v). The drug concentration in the dialysate (C
d
) was determined, and RR
rd
was calculated by the following equation:
$${\text{RR}}_{rd} = \, \left( {C_{p} {-}C_{d} } \right)/C_{p} \times \, 100$$
(4)
In vivo RR was estimated using the retrodialysis-by-drug method. A linear probe was inserted into the dermis of the abdominal skin of a rat anesthetized with intraperitoneal urethane aqueous solution (1.3 g/kg). The rat was perfused with 20% PEG 400 in normal saline (v/v) for 1 h, followed by an equilibration period of 30 min using the STD solution as the perfusate. After a further 30 min of STD perfusion, dialysate samples were collected. UPLC-MS/MS assays were conducted to determine the diffusive loss of the drug indirectly, and RR was calculated using Eq. (4).
In vivo microdialysis studies
To determine the concentration of the administered drug, a probe was inserted into the dermis of the abdominal skin of a rat anesthetized with intraperitoneal urethane aqueous solution (1.3 g/kg), and the active dialysis window was placed below the site of topical drug administration. The rat was immobilized on a mat with the abdomen facing upward and the fur in this region was manually shaven, taking care not to damage the skin. The ambient temperature was maintained at 25°C. For the in vivo microdialysis sampling following probe implantation, the connective tubing from the probe was secured to the skin with adhesive tape to fix its position. The probe was continuously perfused with 20% PEG 400 in normal saline (v/v) at a flow rate of 0.2 mL/h. The skin was allowed to equilibrate for 1 h before a blank sample was taken, and 1 mL of AAS-NLCs or AAS-SLNs was applied 1.5 h after the start of perfusion. A flat cylindrical plastic cover of about 1 cm in height and 1.5 cm in diameter, with an edge width of 2 mm was glued above the application site using cyanoacrylate adhesive, and the drug was applied to the cover the skin just above the probe. During the experiment, the application site, and the probe were kept at the same level. Dialysate samples were collected in small vials, which were replaced every 30 min. Dialysis sampling was performed for 10 h.
Statistical analysis
Results are presented as mean ± standard deviation (SD). Differences were analyzed using the Student’s t test with statistical program for the social sciences (SPSS) software version 19.0 (IBM Corp., Armonk, NY, USA). P values <0.05 were considered to be statistically significant. The uniform design with a U*7(74) table was optimized using the SPSS software and VB software version 6.0 (Microsoft Corp., Redmond, WA, USA). The cutaneous pharmacokinetic parameters of LA and RAN were calculated using noncompartmental analysis with the DAS 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).