Materials & Methods
Materials
Ti foil (99.6%, 0.25 mm thick) supplied by Alfa Aesar (MA, USA) was used as the substrate material for TNT fabrication. TPGS was used for the synthesis of polymer micelles as drug carriers. Indomethacin, an anti-inflammatory water-insoluble drug (with a saturation solubility of 6 mg/ml) was supplied by Sigma-Aldrich (Sydney, Australia). Ethylene glycol and ammonium fluoride (electrolyte), acetone and PBS tablets were also obtained from Sigma-Aldrich. The PBS tablets were reconstituted with 200-ml Milli-Q™ (Millipore, MA, USA) water, 0.0027-M potassium chloride and 0.137-M sodium chloride to yield 0.01-M PBS at pH 7.2 and 20°C. Citrate-functionalized AuNPs were synthesized from their base salts, tetrachloroauric acid trihydrate 99.5% (HAuCl4·3H2O) and trisodium citrate dihydrate (Na3C6O7·2H2O), which were also supplied by Sigma-Aldrich. High-purity water from ultra-pure Milli-Q grade (18.2 MΩcm) was used throughout the study. All chemicals were of analytic reagent grade and used as supplied.
Fabrication of TNTs on Ti as Drug-releasing Implants
TNT arrays on Ti flat foil were prepared by a simple two-step electrochemical anodization process as described previously.[22,42] The substrate was mechanically polished and cleaned by sonication in acetone for 30 min preceding the process. The anodization was performed using ammonium fluoride/ethylene glycol electrolyte, volume 500 ml (3% water/0.3% NH4F/C2H6O2) at 20°C using a constant voltage of 100 V for 2 h in a specially designed electrochemical cell with two electrodes.[4,5] Subsequently, the deposited Ti layer was removed by sonication in methanol and a second anodization was carried out at the same voltage for 1 h to yield an ordered array of TNT on the nanotextured Ti surface.
Preparation of AuNPs
The common approach for reducing gold salt by sodium citrate was applied for obtaining the gold colloidal nanoparticles that were used as stimulants in our DDS.[43] Briefly, 1.2 ml of 0.68-mM sodium tricitrate was added dropwise to a rapidly stirred boiling solution of chloroauric acid (30 ml, 0.3 mM). The gold solution gradually formed as the citrate reduced the gold to Au(III) ions, or Au3+, accompanied by a change in color from yellow to purple, and finally to deep wine red at the end of the reaction in approximately 10 min. The solution was then cooled to room temperature with continuous stirring and characterized thereafter by particle sizing and scanning electron microscopy (SEM).
Synthesis of TPGS Micelles & Drug (Indomethacin) Loading
TPGS polymer micelles were prepared using simplified lyophilization and solvent evaporation techniques.[25,44,45] A total of 5-ml chloroform solution dissolved with 15-mg micelles was stirred under vacuum in a rotary evaporator, followed by dispersing the polymer after solvent evaporation (in a water bath at 70°C) in 20 ml of Milli-Q water. A layer of visible and thin organic micellar film was formed on the wall of the round-bottom flask of the rotary evaporator. Samples were then dialyzed against Milli-Q water for 2 days to produce a homogenized micelle suspension. The solvent was removed under reduced pressure using regenerated cellulose membrane (nominal flat width: 16 mm, diameter: 10 mm and volume/length: 0.79 ml/cm). The loading of drugs in micelles was performed using the same procedure. The drug (indomethacin) and micelles were weighed to prepare 5 wt% of drug relative to micelles. The drug was dissolved in chloroform (30 mg/ml) to make it soluble in the lipid core prior to drying and then added to the 20 vol% micellar solution in Milli-Q water. Drug–micelles (Indomethacin–TPGS) were then dispersed in the Milli-Q water using gentle magnetic stirring for 15 min, followed by continuous and slower stirring to obtain a homogeneous dispersion at room temperature overnight. They were dialyzed against Milli-Q water for 2 days. The solutions were produced in triplicate.
Loading of Drugs & Drug Carriers into TNT–Ti
Prepared TNT–Ti substrates were cut (5 × 5 mm), cleaned using deionized water and dried under nitrogen gas flow. A solution of indomethacin (50 mg/ml) in ethanol and drug-loaded micelle (1% [w/v]) solution were used separately for loading into the TNT–Ti. The substrates were first wetted with ethanol, followed by the addition of eight to ten droplets (1 µl) of AuNP solution. Drug solution (indomethacin) or micelle dispersion (indomethacin–TPGS; 10 µl) was then pipetted onto the TNT layers and spread gently to ensure an even coverage. They were left for 45 min for the drug or drug–micelles to enter the space inside the nanotubes. The surfaces were then allowed to dry in air for 45 min and then under vacuum at room temperature for 1 h for solvent evaporation after addition of each drop of drug/drug–micelle dispersion. The surfaces were wiped using a soft tissue in order to remove excess drug accumulated on top of the surface. The loading, drying and wiping steps were repeated approximately 15–17 times in order to load a substantial amount of drug and drug-loaded micelles inside the TNTs. A ratio of 1:15 (v/v) for AuNPs and drug/drug-loaded micelles was maintained in all sample preparations. All drug-loading and drug-release experiments in this study were performed in triplicate.
Nanomedicine. 2014;9(8):1263-1275. © 2014 Future Medicine Ltd.
